Methods for treating TWEAK-related conditions

ABSTRACT

The present invention provides methods and agents for the treatment of TWEAK-related conditions, including cardiac, liver, kidney, lung, adipose, skeletal, muscle, neuronal, bone and cartilage conditions. The invention also provides methods for identifying TWEAK agonists or antagonists for the treatment of TWEAK-related conditions. Additionally, the invention provides transgenic animals that express an exogenous DNA encoding a TWEAK polypeptide, or fragments, analogs, or muteins thereof, and methods for using such animals to identify TWEAK agonists or antagonists. The invention further provides methods for diagnosing a disease based on TWEAK expression. The invention also provides methods for affecting cellular differentiation of progenitor cells using TWEAK polypeptides, agonists, or antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/179,096, filed Jul. 8, 2011, now abandoned, which is a continuationof U.S. patent application Ser. No. 12/417,755, filed Apr. 3, 2009, nowU.S. Pat. No. 8,506,958, which is a continuation of U.S. patentapplication Ser. No. 10/510,804, filed Sep. 13, 2005, now abandoned,which is a national stage entry of International Application No.PCT/US03/11350, filed Apr. 9, 2003, now published as WO 03/086311 onOct. 23, 2003, and claims the benefit of U.S. Provisional ApplicationNo. 60/371,611, filed Apr. 9, 2002, all of which are incorporated hereinby reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods and agents for the treatment ofTWEAK-related conditions, including cardiac, liver, kidney, lung,adipose, skeletal muscle, neural, bone, cartilage, skin,gastrointestinal, pancreatic, reproductive organ and connective tissuediseases. The invention also relates to methods for identifying TWEAKagonists or antagonists for the treatment of TWEAK-related conditions.Additionally, the invention relates to transgenic animals that expressan exogenous DNA encoding a TWEAK polypeptide, or fragments, analogs, ormuteins thereof and methods for using such animals to identify TWEAKagonists or antagonists. The invention further relates to methods fordiagnosing a disease based on TWEAK expression. The invention alsorelates to methods for affecting cellular proliferation ordifferentiation of progenitor cells using TWEAK polypeptides, agonistsor antagonists.

BACKGROUND OF THE INVENTION

Members of the Tumor Necrosis Factor (TNF) family of ligands, so namedfor their structural similarity to TNF-α, are key components in diverseprocesses, such as inflammatory responses, cellular immunity andapoptosis. TNF ligands may act locally as type II membrane-boundproteins through direct cell-to-cell contact or as secreted proteinshaving autocrine, paracrine or endocrine functions. TNF family membersbind TNF receptor (TNF-R) family members via their C-terminalextracellular domain. Various TNF family members include TNF,lymphotoxins (LT), Fas, CD27, CD30, CD40, 4-1BB, OX-40, TRAMP, CAR-1,TRAIL, GITR, HVEM, osteoprotegrin, NGF, TRAIN, Kay (BAFF), APRIL andTWEAK (TNF relatedness and weak ability to induce cell death).

A defining feature of this family of cytokine receptors is found in thecysteine rich extracellular domain, initially revealed by the molecularcloning of two distinct TNF receptors. This family of genes encodesglycoproteins characteristic of Type I transmembrane proteins having anextracellular ligand binding domain, a single membrane spanning regionand a cytoplasmic region involved in activating cellular functions. Thecysteine-rich ligand binding region exhibits a tightly knit disulfidelinked core domain, which, depending upon the particular family member,is repeated multiple times. Most receptors have four domains, althoughthere may be as few as one, or as many as six.

TNF family members play a role in the regulation of the immune system,controlling cell survival and differentiation, as well as acute hostdefense systems, such as inflammation. Continued efforts in the art tomanipulate members of the TNF family for therapeutic benefit may provideunique means to control disease. For instance, some of the ligands ofthis family can directly induce the apoptotic death of many transformedcells, e.g., LT, TNF, Fas ligand and TRAIL. Fas and possibly TNF andCD30 receptor activation can induce cell death in nontransformedlymphocytes which may display an immunoregulatory function.

The ability to induce programmed cell death is an important andwell-studied feature of several members of the TNF family. Fas mediatedapoptosis appears to play a role in the regulation of autoreactivelymphocytes in the periphery and possibly the thymus. Also, the TNF andCD30 systems have been implicated in the survival of T cells and largecell anaplastic lymphoma lines. Death in this cell line in response toTNF, Fas or LT-β receptor signaling has features of apoptosis.

The TNF family of ligands may be categorized into three groups based ontheir ability to induce cell death. First, TNF, Fas ligand and TRAIL canefficiently induce cell death in many lines and their receptors mostlikely have good canonical death domains. Presumably the ligand to DR-3(TRAMP/WSL-1) would also fall into this category. Next there are thoseligands, such as TWEAK, CD30 ligand, and LTalb2, which trigger a weakerdeath signal limited to a few cells. Studies in these systems havesuggested that a separate weaker death signaling mechanism exists.Lastly, there those members that cannot efficiently deliver a deathsignal. Probably all groups may exert antiproliferative effects on somecell types consequent to inducing cell differentiation, e.g., CD40.

In general, death is triggered following the aggregation of deathdomains which reside on the cytoplasmic side of the TNF receptors. Thedeath domain orchestrates the assembly of various signal transductioncomponents which lead to activation of the caspase cascade. Somereceptors lack canonical death domains, e.g. LTb receptor and CD30, yetcan induce cell death, albeit more weakly. Conversely, signaling throughother pathways such as CD40 is required to maintain cell survival. Thereremains a need to further identify and characterize the functions of theTNF family members, thereby facilitating the development of newtherapies for TNF family-related diseases.

TWEAK was isolated in a screen for RNA that hybridized to anerythropoietin probe. Chicheportiche et al., J. Biol. Chem.272:32401-32410 (1997). The mouse and human peptides have an unusuallyhigh degree of conservation, including 93% amino acid identity in thereceptor binding domain. TWEAK, shown to be efficiently secreted fromcells, is abundantly expressed in many tissues, including heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, lymphnodes, thymus, appendix, and peripheral blood lymphocytes.

One known TWEAK receptor is Fn14, a growth factor-regulatedimmediate-early response gene that decreases cellular adhesion to theextracellular matrix and reduces serum-stimulated growth and migration(Meighan-Mantha et al., J. Biol. Chem. 274:33166-33176 (1999)). Fn14 hasbeen shown to be induced by FGF, calf serum and phorbol ester treatmentand is expressed at relatively high levels in heart, kidney, lung, skin,skeletal muscle, ovary and pancreas tissues, as well as inhepatocellular carcinoma modules and other cancer cell lines, and atlower levels in normal liver tissues.

TWEAK has been implicated in many biological processes. For instance,HT29 cells treated with IFN-γ and TWEAK were shown to undergo apoptosis;although TWEAK's ability to induce apoptosis is weak and only a smallnumber of cell types are susceptible. Chicheportiche et al., J. Biol.Chem. 272:32401-32410 (1997). In contrast, TWEAK has also been shown toinduce angiogenesis and proliferation of endothelial cells in aVEGF-independent pathway. Lynch et al., J. Biol. Chem. 274:8455-8459(1999). Astrocytes are specifically bound and stimulated by TWEAK. TWEAKcan infiltrate an inflamed brain to influence astrocyte behavior.Astrocytes exposed to TWEAK secrete high levels of IL-6 and IL-8, aswell as upregulate ICAM-1 expression. Saas et al., GLIA 32:102-107(2000).

TWEAK has also been implicated in immune system regulation. Uponstimulation with IFN-γ, monocytes rapidly express TWEAK, and anti-TWEAKantibodies partially inhibited their cytotoxic activity against humansquamous carcinoma cells. A combination of anti-TWEAK and anti-TRAILantibodies almost completely inhibited cytotoxicity. Nakayama et al., J.Exp. Med. 192:1373-1379 (2000). In contrast, TWEAK mRNA rapidlydisappeared in mice treated with lipopolysaccharide (LPS), an inducer ofthe immuno-inflammatory responses. Furthermore, TWEAK mRNA was alsoreduced in autoimmune hemolytic anemia and systemic lupus erythematosusin mouse models. These data suggest that the down-regulation of TWEAKexpression is an important event in acute and chronic inflammation.Chicheportiche et al., Biochem. Biophys. Res. Comm. 279:162-165 (2000).

Currently, the art lacks a complete understanding of what conditions ordiseases are related to TWEAK expression and function, including therole of TWEAK in both inflammatory and non-inflammatory conditions.

SUMMARY OF THE INVENTION

The present invention relates to the role of TWEAK in contributing tothe severity and progression of various pathological conditions,including diseases of multiple tissues and organ systems. Suchpathological conditions include acute cardiac injury, chronic heartfailure, non-inflammatory dilated cardiomyopathy, congestive heartfailure, liver epithelial cell hyperplasia, hepatocyte death, liverfibrosis, hepatocyte vacuolation, other liver injuries, bile ductconditions, including bile duct hyperplasia, inflammatory kidneyconditions, such as multifocal inflammation, non-inflammatory kidneyconditions such as tubular nephropathy, tubular hyperplasia, glomerularcysts, glomerular nephropathy, Alport Syndrome, kidney tubularvacuolation, kidney hyaline casts, kidney fibrosis and inflammatory lungdisease. The present invention establishes a causal link between theTWEAK molecule and certain diseases of the heart, liver, kidney andlungs. The invention disclosed herein also establishes a link betweenTWEAK and the behavior of progenitor cells for liver tissue, kidneytubules, skin cells, adipocytes, skeletal muscle, cartilage and bone, aswell as connective tissue cell types, such as stromal cells in the bonemarrow and fibroblasts.

In one embodiment, the invention relates to methods for treatingTWEAK-related conditions, i.e. diseases, settings of injury or otherpathological conditions of tissues wherein a receptor for TWEAK, e.g.FN14, is expressed. Those conditions include fibrosis, cardiomyopathies,and diseases of the kidney, lung, liver, skin, skeletal muscle, lipidmetabolism (e.g. obesity), gastrointestinal tract, pancreas,reproductive organs, neural tissue (including neurodegeneration),cartilage, bone and connective tissue. In a preferred embodiment, theTWEAK-related conditions are non-inflammatory in nature. In anotherpreferred embodiment, the invention relates to methods for treatingTWEAK-related conditions by interfering with the interaction of theTWEAK polypeptide with its cellular receptor.

In other embodiments, the invention relates to TWEAK agonists orantagonists and pharmaceutical compositions comprising them for use intreating TWEAK-related conditions. Such TWEAK agonists or antagonists(i.e. inhibitors) may be anti-TWEAK antibodies, or derivatives thereof;anti-TWEAK receptor antibodies, or derivatives thereof; TWEAKpolypeptide fragments; TWEAK polypeptide analogs; TWEAK muteins; TWEAKmimetics; TWEAK fusion proteins; TWEAK receptor polypeptide fragments;TWEAK receptor polypeptide analogs; TWEAK receptor muteins; TWEAKreceptor mimetics; TWEAK receptor fusion proteins; organic compounds;and inorganic compounds.

In other embodiments, the invention relates to TWEAK agonists orantagonists and pharmaceutical compositions useful in treating hosts inneed of tissue regeneration or replacement. It also relates to use ofTWEAK agonists or antagonists for modulating the behavior of populationsof progenitor cells in vivo or in vitro. The progenitor cells may be theprecursors of liver cell types, kidney tubules, cardiomyocytes, lungcell types, skin cell types, skeletal muscle cell types, adipocytes,gastrointestinal cell types, pancreatic cell types, neural tissue celltypes, cartilage and bone cell types, connective tissue cell types,including stromal cells in the bone marrow and fibroblasts. TWEAKagonists or antagonists and pharmaceutical compositions comprising themmay be administered in vivo to promote tissue regeneration andreplacement in settings of disease or tissue injury, including but notlimited to, toxin, viral, chemotherapy or radiation-induced damage, andgenetic or degenerative disorders. In another embodiment, TWEAK agonistsor antagonists and pharmaceutical compositions thereof could be used incombination with cellular therapy with stem cells or progenitor cells toregenerate tissue and organ systems. In yet another embodiment, stemcells or progenitor cell populations may be expanded in vitro by TWEAKagonists or antagonists and pharmaceutical compositions thereof.Progenitor cell populations expanded through the use of TWEAK agonistsor antagonists may be used for transplantation into hosts in need oftissue regeneration or replacement.

In other embodiments, the invention relates to methods for identifyingTWEAK agonists or antagonists useful as therapeutic agents for thetreatment of TWEAK-related conditions. In another embodiment, theinvention relates to transgenic animals expressing exogenous DNAsencoding TWEAK polypeptides. A further embodiment of this inventionincludes the use of TWEAK as a molecular marker for disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The role of TWEAK in dilated cardiomyopathy is shown. A. AFL-TWEAK transgenic (Tg) mouse shows thrombosis of the right atrium andventricle, as well as severe dilation. B. Normal heart is shown forcomparison.

FIG. 2: TWEAK overexpression in the heart induces cardiac remodeling. Across section of the heart is viewed at 10× magnification withhematoxylin/eosin staining on day 20 following infection of adultC57BL/6 mice with an adenoviral vector comprising murine sTWEAK DNAcompared with an adenovirus-GFP control construct.

FIG. 3: TWEAK induces biliary duct and oval cell hyperplasia, asrevealed in FL-TWEAK transgenic (Tg) mice as compared to non-transgenic(NTg) littermates at 2 weeks of age and 7 months of age.

FIG. 4: TWEAK induces biliary duct and oval cell hyperplasia, asrevealed by increased staining with the A6 mAb which is specific for abiliary epithelial and oval cell marker in FL-TWEAK transgenic (Tg) miceas compared to non-transgenic (NTg) littermates.

FIG. 5: TWEAK induces oval cell hyperplasia as revealed by the presenceof large, oval cells in the portal region in FL-TWEAK transgenic (Tg)mice.

FIG. 6: TWEAK causes hepatocellular vacuolization in FL-TWEAK transgenic(Tg) mice as compared to non-transgenic (NTg) littermates.

FIG. 7: Serum TWEAK levels in mice infected with an adenoviral vectorcomprising murine sTWEAK DNA.

FIG. 8: TWEAK overexpression in the liver induces hepatocyte death andductal hyperplasia. Cross sections of the liver are viewed at 20×magnification with hematoxylin/eosin staining on days 3 and 11 followinginfection of adult C57BL/6 mice with an adenoviral vector comprisingmurine sTWEAK DNA compared with an adenovirus-GFP control construct.

FIG. 9: The TWEAK receptor Fn14 is induced after CCl₄ induced liverinjury in mice. In situ hybridization for Fn14 mRNA in normal mouseliver and CCl₄ induced liver injury shows little if any detectableexpression in normal adult liver and marked induction of Fn14 expressionafter injury. Hemotoxylin and eosin (H&E) stained sections show thecorresponding normal healthy liver and CCl₄ injured liver tissue.

FIG. 10: Fn14 expression is upregulated in biliary epithelial cells in amurine model of bile duct ligation, as revealed by increased stainingwith the anti-sense mRNA probe directed against Fn14 using in situhybridization. Hemotoxylin and eosin (H&E) stained section shows thecorresponding section in bright field microscopy.

FIG. 11: Cross section of FL-TWEAK transgenic (Tg) mouse kidney ascompared to non-transgenic (NTg) mouse kidney at 2 weeks, 8 weeks and 7months of age. The results show tubular basophilia in the TWEAK Tgkidney at 8 weeks and 7 months of age, and dilatation of the urinaryspace in glomeruli, i.e., glomerular cysts, with adjacent basophilictubules at 7 months of age.

FIG. 12: Cross section of FL-TWEAK transgenic (Tg) mouse kidney with H&Estaining. A. Glomerular nephropathy with basophilia of adjacent proximaltubular epithelium is shown. B. Segmental mesangial hypercellularity,hypertrophy of capsular epithelia, and capsular thickening.

FIG. 13: Serial sections from two FL-TWEAK transgenic (Tg) mouse kidneysstained with H&E (top) and proliferating cell nuclear antigen (PCNA)(bottom). Basophilic tubules correspond to tubules expressing PCNA, i.e.proliferating tubules.

FIG. 14: Serial sections from a FL-TWEAK transgenic (Tg) mouse kidneystained with H&E, a lectin from T. Purpureas (a marker for proximaltubules) and a lectin from A. Hypogaea (a marker for distal tubules).Results show that the basophilic tubules do not express eitherepithelial marker.

FIG. 15: TWEAK overexpression in the kidney induces tubular hyperplasiaand glomerulopathy. A cross section of the kidney is viewed at 20× and40× magnification with hematoxylin/eosin staining on day 11 followinginfection of adult C57BL/6 mice with an adenoviral vector comprisingmurine sTWEAK DNA compared with an adenovirus-GFP control construct.

FIG. 16: TWEAK mRNA is widely expressed throughout the kidney in anadult wild-type mouse. A cross section of kidney is viewed at 5×magnification with hematoxylin staining, or under dark field microscopyfollowing in situ hybridization with sense or anti-sense TWEAK probes.

FIG. 17: Fn14 mRNA is expressed in the proximal tubules of outer medullain adult wild-type mouse kidney. A cross section of kidney is viewed at5× magnification with hematoxylin staining, or under dark fieldmicroscopy following in situ hybridization with sense or anti-sense Fn14probes.

FIG. 18: A role for TWEAK in kidney fibrosis is suggested by theupregulation of Fn14 mRNA in Alports kidneys. The fold increase in Fn14mRNA levels is shown in two individual mice carrying the mutationleading to Alports disease relative to wildtype animals at 4, 5, 6, and7 weeks of age. mRNA levels were determined by hybridization to a genechip containing nucleotide sequence corresponding to a portion of theFn14 gene. At the 4 and 7 week time points, replicate results for eachthe two mice are shown (indicated by the mouse 1 repeat and mouse 2repeat bars respectively). At the 7 week time point, Fn14 mRNA is shownto be reduced in two settings where disease is inhibited, i.e. sTGFβR-Fctreatment and in VLA-1 knockout mice (illustrated in FIG. 18 by eitherthe two independent mice treated with sTGFβR-Fc (“sTGFbR treated”) orthe two independent Alport/VLA-1 KO mice (“Alport/VLA-1 KO”).

FIG. 19: TWEAK antagonist treatment in Unilateral Ureteral Obstruction(UUO), a murine model of kidney fibrosis, significantly reduced kidneyfibrosis. Metamorph quantitation of blue-staining area (fibrotic area)on Trichrome-Masson stained paraffin kidney sections indicates thatcollagen content was decreased in AB.G11 (anti-TWEAK monoclonal Ab)treated kidney samples to similar levels observed in sTGF-βR-Ig positivecontrol samples. In contrast, the isotype-control hamster antibody(HA4/8)-treated kidneys showed no reduction in kidney fibrosis, similarto vehicle (PBS)-treated kidneys.

FIG. 20: The TWEAK transgene causes granulomatous and lymphohistocyticinflammation in the lung. A. A cross section of a lung from a FL-TWEAKtransgenic (Tg) mouse with H&E staining. B. A cross section of a lungfrom a sTWEAK Tg mouse with H&E staining.

FIG. 21: TWEAK mRNA is expressed in the cells lining the bronchioles andalveoli in adult wild-type mouse lung. A cross section of lung is viewedat 10× magnification with hematoxylin staining, or under dark fieldmicroscopy following in situ hybridization with sense or anti-senseTWEAK probes.

FIG. 22: Fn14 mRNA is expressed in the bronchioles and alveoli of adultwild-type mouse lung. A cross section of lung is viewed at 10×magnification with hematoxylin staining, or under dark field microscopyfollowing in situ hybridization with sense or anti-sense Fn14 probes.

FIG. 23: Inhibitory effect of TWEAK on 3T3-L1 cell adipocytedifferentiation in vitro. 3T3-L1 cells were induced to undergodifferentiation using a standard protocol. Cells were untreated, treatedwith a control agent (recombinant soluble human CD40L-FLAG 100 ng/ml) orvarious versions of TWEAK at 100 ng/ml recombinant soluble humanTWEAK-FLAG, recombinant soluble human TWEAK, Fc-human TWEAK) on day 0,together with dexamethasone and insulin, and were replenished daily. Inone experimental group, the blocking anti-TWEAK mAb AB.G11 was alsoadded at the same time as Fc-hTWEAK. The cells were stained with Oil-redO on day 7.

FIG. 24: Inhibitory effect of TWEAK on myogenesis in vitro. C2C12myoblasts were grown to near confluency in a DMEM-based growth media andon day 0, switched to a low-serum differentiation media that contained2% horse serum to trigger differentiation. Cells were untreated ortreated on day 0 with Fc-hTWEAK (100 ng/ml). Myotube formation wasexamined using a phase-contrast microscope and pictures were taken onday 6 of differentiation. In other experimental groups, Fn14-Fc or aneutralizing anti-TNF antibody were added at the same time as Fc-hTWEAK,thereby demonstrating that the inhibitory effect of Fc-hTWEAK wasTWEAK-specific and not mediated through TNF.

FIG. 25: TWEAK can bind to human mesenchymal stem cells. Humanmesenchymal stem cells (hMSCs) were incubated with recombinant Fc-TWEAKprotein followed by PE-conjugated goat anti-human Fc or goat anti-mouseFc secondary antibodies. The ability of Fc-TWEAK to bind to hMSCs wasdetermined using fluorescence activated cell sorter (FACS) analysis. Thebackground staining is provided by the secondary antibody staining (2ndonly) alone.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, genetics,virology and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following terms are employed:

“Antibody” refers to an intact immunoglobulin, or to an antigen-bindingportion thereof that competes with the intact antibody for specificbinding. Antigen-binding portions may be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies.Antigen-binding portions include, inter alia, Fab, Fab′, F(ab′)2, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), chimeric antibodies, diabodies andpolypeptides that contain at least a portion of an immunoglobulin thatis sufficient to confer specific antigen binding to the polypeptide. AnFab fragment is a monovalent fragment consisting of the VL, VH, CL andCH1 domains; a F(ab′)2 fragment is a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consists of the VH and CH1 domains; an Fv fragment consists ofthe VL and VH domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546, 1989) consists of a VH domain. Asingle-chain antibody (scFv) is an antibody in which a VL and VH regionsare paired to form a monovalent molecules via a synthetic linker thatenables them to be made, as a single protein chain (Bird et al., Science242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883, (1988)). Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993), and Poljak et al., Structure 2:1121-1123 (1994)).One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an immunoadhesin. An immunoadhesin mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody hastwo different binding sites.

“Antibody repertoire” refers to the sum of every different antibodyspecies in an animal or human. Diversity in antibody repertoires resultsfrom, inter alia, immunoglobulin gene recombination, immunoglobulin genejunctional diversity, terminal deoxytransferase activity, and somatichypermutation.

“Chimeric antibodies” are antibodies that have been altered from theiroriginal form to comprise amino acid sequences from another protein.Chimeric antibodies retain at least a portion of the original antibodyamino acid sequence, typically the portion comprising the antigenbinding region (Fab). Examples of chimeric antibodies include, but arenot limited to, bispecific antibodies and fusions with othernon-immunoglobulin protein sequences.

“cis regulatory elements” generally refer to sequences that regulate theinducible or constitutive expression of gene sequences under specificconditions or in specific cells. Examples of cellular processes thatexpression control sequences regulate include, but are not limited to,gene transcription, protein translation, messenger RNA splicing,immunoglobulin isotype switching, protein glycosylation, proteincleavage, protein secretion, intracellular protein localization andextracellular protein homing.

“Cytokines” refer generally to signaling molecules of the immune system.Cytokines include, but are not limited to, Interleukins (IL),transforming growth factors (TGF), tumor necrosis factors (TNF),lymphotoxins (LT), interferons, granulocyte-macrophage colonystimulating factors (GM-CSF), macrophage CSF, Granulocyte CSF, andmigration inhibition factors.

“Embryonic stem (ES) cells” refer to pluripotent or multipotent cellsthat can, when injected into a blastocyst, contribute to many or alltissues of a prenatal, postnatal or adult animal. Animals that resultfrom blastocyst injections are often referred to as “chimeric” animalssince their somatic and/or germ cells are often derived from both theblastocyst donors and the injected ES cells. One important property ofES cells is their ability to contribute to the germ line of the animals,resulting in any desired heritable characteristics to be passed to thechimeric animal's progeny. Immortalized ES cells are a powerful tool forgenerating animals with targeted disruptions of endogenous genesequences or for generating animals with foreign genes (transgenes).

“Expression control sequences” refer to sequences that allow for theconstitutive or inducible expression of gene sequences under specificconditions or in specific cells. Examples of cellular processes thatexpression control sequences regulate include, but are not limited to,gene transcription, protein translation, messenger RNA splicing,immunoglobulin isotype switching, protein glycosylation, proteincleavage, protein secretion, intracellular protein localization andextracellular protein homing.

“Fusion Proteins” refer to chimeric proteins comprising amino acidsequences of two or more different proteins. Typically, fusion proteinsresult from in vitro recombinatory techniques well known in the art.However, fusion proteins may result from in vivo crossover or otherrecombinatory events.

“Human immunoglobulin molecules” refer to immunoglobulin proteins thatare encoded by human immunoglobulin gene sequences. The immunoglobulingene sequences may be expressed in any cell or animal, human ornon-human.

“Humanized antibodies” are antibodies that are derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans. Alternatively, humanizedantibodies may be produced by fusing the constant domains from a humanantibody to the variable domains of a non-human species. Examples of howto make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

“Inflammation” or “inflammatory disease” refers to the fundamentalpathologic process consisting of cytologic and histologic reactions thatoccur in blood vessels and adjacent tissues in response to injury,abnormal stimulation or biological agents. Likewise, “non-inflammatoryconditions” or “non-inflammatory diseases” refer to any condition ordisease that is not inflammatory in nature, as disclosed herein.

“Isolated protein” or “isolated polypeptide” refers generally to aprotein or polypeptide that by virtue of its origin or source ofderivation: (1) is not associated with naturally associated componentsthat accompany it in its native state, (2) is free of other proteinsfrom the same species, (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature. Thus, a polypeptide that ischemically synthesized, synthesized in a cell-free biological system(e.g., a rabbit reticulocyte lysate), or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A protein mayalso be rendered substantially free of naturally associated componentsby isolation, using protein purification techniques well known in theart.

“Mimetics” or “peptide mimetics” are non-peptide analogs that arecommonly used in the pharmaceutical industry as drugs with propertiesanalogous to those of the template peptide. Fauchere, J. Adv. Drug Res.15:29 (1986); Veber and Freidinger, TINS p. 392 (1985); and Evans etal., J. Med. Chem. 30:1229 (1987), incorporated herein by reference.Mimetics are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, mimetics are structurallysimilar to a paradigm polypeptide (i.e., a polypeptide that has adesired biochemical property or pharmacological activity), such asTWEAK, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH2NH—, —CH2S—,—CH2-CH2-, —CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and —CH2SO—,by methods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

“Polypeptide analogs” refer to polypeptides that are derived fromwild-type polypeptides but differ therefrom in their amino acidsequences. Polypeptides with changes in their amino acid sequences maybe muteins, fusion proteins, or mimetics. Polypeptide analogs also referto polypeptides that have non-amino acid sequence differences ascompared with the wild-type polypeptides. These differences may bechemical or biochemical, and include, but are not limited to, the typesof modifications specifically disclosed herein.

“Polypeptide fragments” refer to polypeptides that have anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thenaturally-occurring sequence. Fragments typically are at least 5, 6, 8or 10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long.

“Progenitor cells” refer to cells that can give rise to one or more celllineages. Included are stem cells, totipotent cells, pluripotent cells,multipotent cells, bipotent cells, embryonic cells or adult cells. Alsoincluded are tissue-specific cells, including, but not limited to, cellscommitted to a particular lineage capable of undergoing terminaldifferentiation, cells that derive from tissue resident cells, andcirculating cells that have homed to specific tissues.

“Subjects” are humans and non-human subjects. An example of a subject isa patient.

“TWEAK-related conditions” refer to any conditions that result fromaberrant TWEAK function or regulation. The term may also refer to anycondition that does not directly result from aberrant TWEAK function orregulation, but rather arises out of some other mechanism whereindisrupting, increasing or otherwise altering TWEAK activity will have adetectable outcome on the condition. TWEAK-related conditions can beeither inflammatory or non-inflammatory in nature, and include, but arenot limited to, the conditions and diseases specifically disclosedherein.

“Vectors” refer to DNA molecules that allow DNA sequences of interest tobe cloned, propagated, recombined, mutated, or expressed outside oftheir native cells. Often vectors have expression control sequences thatallow for the inducible or constitutive expression of gene sequencesunder specific conditions or in specific cells. Examples of vectorsinclude, but are not limited to, plasmids, yeast artificial chromosomes(YACs), viruses, Epstein Bar Virus (EBV)-derived episomes,bacteriophages, cosmids and phagemids.

“Xenogeneic animals” refer to animals bearing substantial portions ofhuman immunoglobulin loci. Often, xenogeneic animals bear homologouslytargeted endogenous immunoglobulin loci, rendering them incapable ofexpressing their endogenous immunoglobulin genes. Examples include themice of the XenoMouse™ line (Abgenix, Inc., Fremont, Calif.), which arecapable of somatic rearrangement of transgenic human immunoglobulingenes, hypermutation of the human variable genes, immunoglobulin geneexpression, and immunoglobulin isotype switching. Xenogeneic animals arecapable of mounting effective humoral responses to antigenic challengeutilizing the human immunoglobulin gene sequences. Antibodies producedin xenogeneic animals are fully human and can be isolated from theanimals themselves or progeny thereof, from cultured cells extractedfrom the animals or progeny thereof, and from hybridomas created fromxenogeneic B lymphocytic lines or progeny thereof. Moreover, therearranged human gene sequences encoding immunoglobulins raised againstspecific antigenic challenges can be isolated by conventionalrecombinant techniques.

“Xenogeneic antibodies” refer to antibodies that are encoded by foreignimmunoglobulin loci. For example, in mice of the XenoMouse™ line, thehuman antibody loci encode xenogeneic antibodies.

“Xenogeneic monoclonal antibodies” refer to homogenous populations ofantibodies that are produced in cloned, immortalized cells, e.g.hybridomas, derived from xenogeneic animals. For example, hybridomasmade from mice of the XenoMouse™ line produce xenogeneic antibodies.

The understanding and treatment of diseases fundamentally advances upondetermination of the molecular mechanisms or biochemical pathwaysunderlying them. Physicians and researchers are thereby enabled totailor therapeutic agents and formulate pharmaceutical compositions thatspecifically target those molecular mechanisms or biochemical pathways.

Some of the most complex and debilitating diseases afflicting humansinclude those of the heart, liver, kidney, lung, skin, skeletal muscle,lipid metabolism, gastrointestinal tract, nervous system, pancreas,reproductive organs, cartilage, bone, connective tissue system, andprogenitor or stem cells. The present invention advantageously providesimportant advances in the understanding of these diseases. Moreparticularly, the invention provides transgenic animals which expressexogenous TWEAK proteins and demonstrate for the first time acorrelation between expression of TWEAK protein and certain pathologicalconditions of the heart, liver, kidney and lung. The invention alsoprovides methods for treating or preventing such pathologicalconditions, as well as methods for identifying TWEAK agonists orantagonists for use in those methods. Pathological conditions that maybe treated according to the methods of this invention include acutecardiac injury, chronic heart failure, non-inflammatory dilatedcardiomyopathy, congestive heart failure, liver epithelial cellhyperplasia, hepatocyte death, liver fibrosis, hepatocyte vacuolation,liver injury, bile duct conditions, including bile duct hyperplasia,inflammatory kidney conditions, such as renal multifocal inflammation,non-inflammatory kidney conditions, such as tubular nephropathy, tubularhyperplasia, glomerular cysts, renal hyperplasia, renal capsularthickening, glomerular nephropathy, Alport Syndrome, kidney tubularvacuolation, kidney hyaline casts, kidney fibrosis and inflammatory lungconditions. The invention further provides methods for detecting TWEAKstructures or functions as molecular markers of disease, including TWEAKproteins or their functions, TWEAK antibodies and TWEAK nucleic acids.

The TWEAK-related conditions described herein are treated using TWEAKagonists or antagonists that are capable of altering TWEAK activity ordisrupting the interaction between a membrane-bound or full-length formof TWEAK polypeptides with its cellular receptors. Alternatively, thetherapeutic agents and treatment methods disrupt the interaction betweena membrane-bound or full-length form of TWEAK polypeptides with anotherTWEAK polypeptide. Such interference may occur on the surface of a cell,intra-cellularly, extra-cellularly, or in vitro bound to a solid phaseor in solution. In another alternative, the therapeutic agents andtreatment methods disrupt the interaction between membrane-bound orfull-length forms of TWEAK polypeptides and TWEAK interacting partners.Such interacting partners may be proteins, nucleic acids, saccharides,lipids, fatty acids, and steroids.

In a preferred embodiment of this invention, the TWEAK-related conditionis non-inflammatory in nature.

In another preferred embodiment, the TWEAK-related condition isfibrosis, cardiomyopathy, kidney disease, lung disease or liver disease.

In another preferred embodiment, the TWEAK-related condition is skeletalmuscle disease, adipose tissue disease, gastrointestinal tract disease,pancreatic disease, a reproductive organ disease, a neural tissuedisease, cellular death, skin disease, cartilage disease, bone disease,or connective tissue disease.

In another embodiment, TWEAK agonists or antagonists may be used totreat subjects suffering from a condition, disease or injury thatrequires tissue replacement or regeneration (e.g. burn victims orradiation patients) by affecting progenitor cells in vivo. The TWEAKagonists or antagonists may also be used to treat subjects in vivo incombination with progenitor cell or tissue transplantation therapy. TheTWEAK agonists or antagonists may also be used to expand cellpopulations in vivo or progenitor cell populations in vitro forsubsequent transplantation into subjects with or without additionaltreatment. Progenitor cell populations used for in vivo cell therapy orin vitro expansion followed by transplantation may be embryonic or adultin origin. Adult-derived progenitors may be multipotent ortissue-restricted (Lagasse et al., Immunity 14:425-436 (2001); Jacksonet al. J. Clin. Invest. 107:1355-402 (2001); Anversa and Nadal-Ginard,Nature 415:240-243 (2002); Gussoni et al., Nature 401:390-394 (1999);Brazelton et al., Science 290:1672-1674 (2000); Peterson et al., Science284:1168-1170 (1999); Lagasse et al., Nature Medicine 6:1229-1234(2000)).

Heart disease is the predominant cause of disability and death inindustrialized nations. In the United States, heart disease causes about40% of all mortalities, accounting for approximately 750,000 deathsannually. Most basic to the function of the heart is the myocardium,composed primarily of branching and anastomosing striated muscle cells(cardiac myocytes). Cardiac myocytes are much larger than theintervening interstitial cells, accounting for more than 90% of thevolume of the myocardium. Inflammatory cells are rare and collagen issparse in a normal myocardium.

Myocardial disease is common but occurs secondarily in a number ofdifferent heart conditions. Examples of myocardial disease includeinflammatory disorders (e.g., myocarditis), and non-inflammatory heartconditions such as dilated cardiomyopathy, systemic metabolic disorders,muscular dystrophies, and genetic abnormalities in cardiac muscle cells.

The major types of cardiomyopathy include dilated, hypertrophic andrestrictive cardiomyopathies. It is an object of the invention toprovide methods for the treatment of dilated cardiomyopathy, which istypically non-inflammatory in nature. In the case of non-inflammatorydilated cardiomyopathy, which accounts for approximately 90% of theclinical cases of myocardial disease, the heart is characterized byprogressive cardiac hypertrophy, dilation, and contractile (systolic)dysfunction. Dilated cardiomyopathy may occur at any age, but is mostcommon in persons ranging in age from 20 to 60 years old. Diagnosis isoften made through noninvasive cardiac imaging, particularly throughtwo-dimensional echocardiography. The histopathology of dilatedcardiomyopathy is characterized by degenerating myocytes with mild tomoderate hypertrophy, an absence of inflammatory cells, and interstitialfibrosis.

Clinically, dilated cardiomyopathy presents with slowly progressivecongestive heart failure, but patients may slip precipitously from acompensated to a decompensated functional state. Cardiac transplantationis frequently required. Fifty percent of patients die within two years,and seventy five percent within five years. The cause of death istypically progressive cardiac failure or arrhythmia, however, embolismcaused by dislodgment of an intracardiac thrombus may occur.

Hearts characterized by dilated cardiomyopathy are enlarged, flabby, andweigh two to three times as much as normal hearts. All chambers aredilated, with wall thinning, fibrosis, and typically mural thrombi. In aminority of dilated cardiomyopathies, mitral or tricuspid regurgitationresults from left ventricular chamber dilation. Cardiac muscle cells arehypertrophied with enlarged nuclei. Some of the causes of dilatedcardiomyopathy include myocarditis, alcohol or other toxin abuse,pregnancy (peripartum cardiomyopathy), ischemia, coronary arterydisease, hypertension, and genetic influences.

Idiopathic Dilated Cardiomyopathy (IDC), a disease of unknown etiology,is characterized by dilation of one or both ventricles, systolicdysfunction, and often progression to congestive heart failure. It isnoted that the term “IDC” is used by some persons of skill in the artinterchangeably with the term “dilated cardiomyopathy”, suggesting thatIDC is in fact a broad category of dilated cardiomyopathies that are notthe result of alcohol abuse, toxic insult or myocarditis.

Microscopically, IDC is characterized by myocardiocyte hypertrophy,karymegaly and interstitial and perivascular fibrosis. In contrast tomyocarditis, necrosis and cellular infiltration are not typicallyprominent in IDC patients, an indication of its non-inflammatoryetiology. Consistent with that etiology is the fact thatanti-inflammatory drugs, such as prednisone, are largely ineffective intreating IDC.

It is an object of this invention to provide methods of treating orpreventing dilated cardiomyopathy associated with TWEAK activity.Because the cause of dilated cardiomyopathy (e.g., IDC) is largelyunknown, specific therapies have not been developed. Patients aretypically treated for heart failure using physical, dietary, andpharmacological interventions (e.g., beta-adrenoceptor antagonists,calcium antagonists, and anticoagulants) to control the symptoms. Also,cardiac transplantation is used when available.

Those of skill in the art have been unable to identify anyimmunological, histochemical, morphological, ultrastructural ormicrobiological marker that might be used to diagnose IDC. However,epidemiological evidence suggests that predisposition to IDC may begenetically-based. In 20 percent of IDC patients, a first-degreerelative also shows evidence of IDC, suggesting frequent familialtransmission. Those of skill in the art have recognized the need todetermine molecular genetic markers for IDC in subclinical and clinicalheart disease patients.

To date, the list of genes associated with dilated cardiomyopathyincludes cardiac troponin T, d-sarcoglycan, cardiac b myosin heavychain, cardiac actin, a-tropomyosin, Lamin A/C, Desmin, cardiacryanodine receptor, desmoplakin, plakoglobin, dystrophin, and tafazzin.The need still exists to find additional genetic factors that contributeto dilated cardiomyopathy and to design therapeutics that target them.The present invention has, for the first time, demonstrated a causallink between TWEAK and dilated cardiomyopathy. It is therefore an objectof the invention to provide a method for identifying dilatedcardiomyopathy in a patient by detecting changes in TWEAK proteinexpression, TWEAK protein function, TWEAK mRNA expression, or achromosomal alteration. Methods and reagents for detecting suchmolecular markers of disease are well known in the art, and involveimmunological or immunohistochemical analyses, enzyme or otherprotein-function assays, and standard hybridization-based assays such asnorthern blots, Southern blots, single nucleotide polymorphism (SNP)analysis, and fluorescence in situ hybridization (FISH) analysis.

It should be noted that non-inflammatory dilated cardiomyopathy ischaracterized by progressive cardiac hypertrophy, dilation, andcontractile dysfunction. In contrast, Chagas' disease is a rare form ofmyocarditis, which is an inflammatory heart disease that develops inhumans and experimental animals following chronic Trypanosoma cruziinfection. Studies of Chagas' disease, which is prevalent in Central andSouth America, have identified anti-self antibodies in the sera ofChagas' disease patients. Joshua Wynne and Eugene Braunwald, HeartDisease, A Textbook of Cardiovascular Medicine, Volume 2, Ch. 41, pp.1442-1444 (5th ed. 1997). The methods disclosed herein are directed tothe treatment of the more common, non-inflammatory-type dilatedcardiomyopathy associated with TWEAK activity.

An adult human kidney processes more than 1,700 liters of blood per day,resulting in approximately 1 liter of urine. The kidney functions inwaste excrement, metabolism, water, salt and pH homeostasis, as well ascontributing to the endocrine system. Renal diseases are more likely tocause morbidity than mortality, with approximately 35,000 deathsannually in the United States. In contrast, millions of persons areafflicted annually by non-fatal kidney diseases such as infections,kidney stones, and urinary obstruction.

Typically, glomerular diseases are caused by immunological disorders,whereas tubular and interstitial disorders are usually caused by toxinsor infectious agents. A partial list of kidney diseases includes acutenephritic syndrome, asymptomatic hematuria or proteinuria, acute renalfailure, chronic renal failure, renal tubular defects, urinary tractinfection, nephrolithiasis, urinary tract obstruction and kidneyfibrosis.

Kidney injuries that involve the tubules typically involve theinterstitium as well. Diseases of the tubules can be inflammatory ornon-inflammatory in nature and include ischemic or toxic tubular injury.A partial list of tubule diseases includes acute tubular necrosis andacute renal failure; inflammatory reactions of the tubules andinterstitium (tubulointerstitial nephritis); tubular hyperplasia;tubulointerstitial fibrosis (a scarring disease initiated by tubularepithelial cells with interstitial fibroblast, mononuclear cell,glomerular ultrafiltrate, cytokine and chemokine components); andautosomal dominant polycystic kidney disease (ADPKD) (an inheriteddisorder characterized by large, fluid-filled cysts from the tubules andCollecting ducts and caused by a mutation in either the PKD1 or PKD2genes).

Glomerular diseases represent the most daunting of the kidney diseases.For instance, chronic glomerulonephritis is the most common cause ofchronic renal failure in humans. In the so-called secondary glomerulardiseases, glomeruli may be injured by immunologic disorders such assystemic lupus erythematosus (SLE), as well as vascular disorders, e.g.,hypertension and polyarteritis nodosa. Also, metabolic diseases, such asdiabetes mellitus (i.e. diabetic nephropathy) and hereditary conditions,such as Fabry disease, affect the glomeruli. The primary glomerulardiseases include primary glomerulonephritis and glomerulopathy.

The group of diseases under the umbrella of hereditary nepthritisincludes familial renal diseases associated primarily with glomerularinjury. Alports syndrome is a form of nephritis that is accompanied bynerve deafness and various eye disorders, including lens dislocation,posterior cataracts, and corneal dystrophy. The disease is moreprevalent in males because of its dominant X-linked genotype. However,some females are afflicted due to one of either an autosomal dominantand recessive genotype. The glomeruli of Alport kidneys show segmentalproliferation or sclerosis. Sometimes the epithelial cells acquire afoamy appearance, due to neutral fat and mucopolysaccharideaccumulation. The glomular and tubular basement membranes show irregularfoci of thickening or attenuation, with splitting and lamination of thelamina densa.

Because kidney diseases are of significant clinical importance, those ofskill in the art have recognized the need to understand theirphysiological and genetic causes. Skilled artisans further recognize theneed to develop new therapeutic agents for treatment of chronic andacute kidney diseases. The invention demonstrates for the first time acausal link between TWEAK and kidney disease.

Therefore, in one embodiment, the invention provides methods for thetreatment of kidney diseases. In a more preferred embodiment, the kidneydisease may be Alport syndrome. In other more preferred embodiments ofthe invention, the target kidney diseases may be characterized bymultifocal inflammation, tubular nephropathy, tubular hyperplasia,cysts, glomerular nephropathy, tubular vacuolation, fibrosis or hyalinecasts.

Lung disease has been, and remains, a prevalent affliction. Primaryrespiratory infections, such as bronchitis, bronchopneumonia and othertypes of pneumonia, must commonly be treated by clinicians. Lungdiseases may be exacerbated by environmental factors such as cigarettesmoke, air pollution and other inhalants. Chronic bronchitis, emphysema,pulmonary fibrosis and malignancy are also quite common. The lungs arealso secondarily involved in many terminal diseases, with pulmonaryedema, atelectasis, or bronchopneumonia found in most critically-illpatients.

Asthma is a chronic relapsing inflammatory disorder characterized byhyper-reactive airways. The symptoms are typically characterized by anepisodic, reversible bronchoconstriction. Asthma is caused by anincreased responsiveness of the tracheobronchial tree to various stimuliand is often associated with an IgE response to external allergens.

There are two major types of asthma. The first type is extrinsic asthma,which is initiated by a type hypersensitivity reaction induced byexposure to an extrinsic antigen. The list of extrinsic asthmaconditions includes atopic (allergic) asthma, occupational asthma, andallergic bronchopulmonary aspergillosis. The second type is intrinsicasthma, which results from nonimmune mechanisms, and is triggered byfactors, such as aspirin ingestion, pulmonary infections, stress, cold,inhaled irritants, and exercise.

Those of skill in the art have recognized the need for a betterunderstanding of lung disease, including both non-inflammatory andinflammation-based diseases, such as asthma. An increased understandingwill facilitate the development of improved pharmaceutical agents fortreating lung diseases. The present invention demonstrates for the firsttime a causal link between TWEAK and lung disease, and methods oftreatment thereof. In more preferred embodiments of this invention, thelung disease is characterized by inflammation, including granulomatousand/or lymphohistiocytic inflammation.

The liver is the primary regulator of digestion and metabolichomeostasis, including the processing of dietary amino acids,carbohydrates, lipids, and vitamins, as well as the synthesis of serumproteins, detoxification, and excretion into the bile of endogenouswaste products and pollutant xenobiotics. Thus, hepatic disease istypically very serious, sometimes life-threatening.

The liver is vulnerable to many types of diseases, including metabolic,toxic, microbial, circulatory, and neoplastic insults. Toxins orimmunological disorders may cause hepatocytes to swell, and becomeedematous in appearance, with irregularly clumped cytoplasm and largeclear spaces. Also, retained biliary material may cause the hepatocytesto become foamy and swollen. Accumulation of substances such as iron,copper and fat droplets (steatosis) can accumulate in hepatocytes. Incases of alcoholic liver disease and acute fatty liver of pregnancy,tiny droplets that do not displace the nucleus appear (known asmicrovesicular steatosis).

Hepatocyte necrosis, which results from significant liver injury, can becharacterized by, inter alia, ischemic coagulative necrosis. Often, celldeath from toxic or immunologically related conditions is characterizedby rounded up hepatocytes and shrunken, pyknotic, intensely eosinophilic“Councilman bodies” containing fragmented nuclei (resulting fromapoptosis). Alternatively, hepatocytes may become osmotically swollenand rupture (lytic necrosis).

Hepatitis results from some injury to the liver associated with aninflux of acute or chronic inflammatory cells. Hepatocyte necrosis mayprecede the onset of inflammation, or vice versa. Fibrosis, anirreversible consequence of hepatic damage, usually results frominflammation or non-inflammatory mechanisms, such as a direct toxicinsult. The characteristic deposition of collagen affects blood flow andperfusion of hepatocytes. With continuing fibrosis, the liver subdividesinto nodules of regenerating hepatocytes with surrounding scar tissue(cirrhosis).

Oval cells are so named because of their morphological appearance assmall, proliferating epithelial cells with an ovoid nucleus and scantbasophilic cytoplasm. Oval cells normally reside in the terminal bileductules that connect the intrahepatic ducts which are located in theportal triad with the hepatocyte cords. These cells may be derived fromresident liver progenitor cells, or from bone marrow progenitor cellsthat have circulated and homed to the liver. These ductular progenitorcells have the potential to differentiate into both bile duct epithelialcells and hepatocytes. The present invention demonstrates thatexpression of a TWEAK transgene in mice has the capacity to expand thepopulation of ductular progenitor cells.

Because of the high levels of morbidity and mortality caused by liverdisease, the art has recognized that the molecular and geneticunderpinnings of that disease must be elucidated. Identification ofcausative factors facilitates the discovery of therapeutic agents fortreatment of chronic and acute liver diseases. The present inventiondemonstrates a causal link between TWEAK and liver disease, andadvantageously provides methods for the treatment thereof. In a morepreferred embodiment, the liver disease is epithelial hyperplasia,hepatocyte vacuolation, bile duct injury resulting in fibrosis,hepatocyte death or liver injury.

The skeletomuscular system is critical for posture and locomotion.Skeletal muscle can atrophy in response to disuse, which may besecondary to conditions of nerve or blood supply deprivation and drugexposure such as glucocorticoids. Skeletal muscle can also atrophy inconditions of genetic or degenerative disorders. These conditions ordiseases can be inflammatory or noninflammatory in nature. Musculardystrophy constitutes a large group of hereditary myopathiescharacterized by atrophy and loss of muscle fibers in the absence ofnerve disease; one common form that is included in this group isDuchenne's muscular dystrophy. Congenital muscle disease may also occurin the context of glycogen storage diseases, such as acid maltasedeficiency, which results in babies with weak muscles, poor athletes,enlarged hearts, and often early death from cardiac failure. Congenitaldisorders leading to muscle atrophy also include, but are not limitedto, mitochondrial myopathies, lipid myopathies, central tubularmyopathies, and rhabdomyolysis. Myopathic conditions also may develop inadults, one of the most commonly observed being alcoholic myopathy.Skeletal muscle wasting also may occur as a component of neuronaldisease, including but not limited to, amyotrophic lateral sclerosis(ALS). In addition, skeletal muscle wasting, also known as cachexia, isan important pathological condition seen in most terminally ill cancerpatients and often is directly responsible for patients' death. Diseasesof skeletal muscle that occur in the context of inflammation orautoimmunity include polymyositis, inflammatory myopathies, andglucocorticoid induced atrophy. The present invention establishes a linkbetween TWEAK and the ability of myoblasts to differentiate intomyotubes. It is therefore an object of the invention to provide methodsof treatment of skeletal muscle disorders by promoting skeletal muscleregeneration using in vivo or in vitro approaches.

Accumulation of fat cells occurs in conditions of obesity, includingobesity associated with metabolic disorders such as Type II diabetes.Ingrowth into organs of fat cells, so-called fatty infiltration, occursin a variety of settings, and is a pathological component of musculardystrophies. The present invention has demonstrated a link between TWEAKand the ability of preadipocytes to differentiate into adipocytes. It istherefore an object of the invention to provide methods of treatment ofdisorders associated with an accumulation or paucity of adipocytes bymodulating adipocyte differentiation with TWEAK agonists or antagonistsor pharmaceutical compositions thereof.

The methods of treating a TWEAK-related condition according to thepresent invention utilize TWEAK agonists or antagonists or compositionscomprising them. TWEAK agonists or antagonists useful in treatingTWEAK-related conditions according to this invention are describedherein and are known in the art. Such agents include those disclosed in,e.g., PCT International Publication Nos. WO 98/05783, WO 98/35061, WO99/19490, WO 00/42073, and WO 01/45730, all of which are incorporatedherein by reference. TWEAK antagonists useful in the methods of theinvention include anti-TWEAK antibodies, such as antibodies that arehuman, non-human, humanized or xenogeneic, as described herein, and arepolyclonal, monoclonal, or synthetic. Furthermore, the antibodies may befull-length, fragments thereof, or fusion proteins that include antigenrecognition sequences.

TWEAK antagonists useful in the methods of the invention also includeanti-TWEAK receptor antibodies. Here, the TWEAK receptor may be FN14 orother members of the TNF-R family that are bound by TWEAK. Theantibodies to the TWEAK receptor may be human, non-human, humanized orxenogeneic, as described herein, and are polyclonal, monoclonal, orsynthetic. Furthermore, the antibodies may be full-length, fragmentsthereof, or fusion proteins that include antigen recognition sequences.

Immunization of animals with TWEAK or TWEAK receptor antigens may becarried out by any method known in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press,1990. Methods for immunizing non-human animals, such as mice, rats,sheep, goats, pigs, cattle, horses and the like are well known in theart. See, e.g., Harlow and Lane and U.S. Pat. No. 5,994,619. In apreferred embodiment, the antigen is administered with or without anadjuvant to stimulate the immune response. Such adjuvants include, interalia, complete or incomplete Freund's adjuvant, RIBI (muramyldipeptides) or ISCOM (immunostimulating complexes).

The antigen chosen for immunization can be any one of the following: aTWEAK polypeptide; a TWEAK polypeptide fragment; a TWEAK mutein; a TWEAKmimetic; a TWEAK fusion protein; a TWEAK receptor polypeptide; a TWEAKreceptor fragment; a TWEAK receptor mutein; a TWEAK receptor mimetic; aTWEAK receptor fusion protein; a cell expressing a TWEAK polypeptide,fragment, mutein, or fusion protein thereof; or a cell expressing aTWEAK receptor polypeptide, fragment, mutein, or fusion protein thereof.The immunoglobulins raised in the animals by immunization may berecovered from various tissues or fluids of the animals, includingserum, milk, ascites, spleen, thymus, peripheral blood cells, fetalliver, bone marrow, peritoneum, and any other tissues or fluids havingsignificant immunoglobulin concentrations. Also, hybridomas can be madeand isolated that produce monoclonal antibodies secreted into themedium.

In preferred embodiments of this invention, the antibodies arepolyclonal antibodies, monoclonal antibodies, or humanized antibodies.In a more preferred embodiment, the humanized antibodies comprise humanantibody constant and/or framework regions. In another preferredembodiment, the antibodies are xenogeneic antibodies. In more preferredembodiments, the xenogeneic antibodies are polyclonal antibodies ormonoclonal antibodies.

Xenogeneic antibodies are complete antibodies of one species that areexpressed in an entirely different species. For instance, if a mouseexpresses the DNA required to produce complete human antibodies, theresulting antibodies are xenogeneic (i.e. human antibodies produced in amouse). Targeted inactivation (knockout) technology provides theopportunity to disrupt an animal's normal expression of endogenousimmunoglobulin genes. Transgenic animal technology provides theopportunity to produce non-human animals that produce xenogeneicimmunoglobulin proteins. Such xenogeneic animals can be mated to theimmunoglobulin knockout animals described above, resulting in animalsthat produce only the xenogeneic immunoglobulins and not endogenousimmunoglobulins.

Expression of xenogeneic immunoglobulin genes allows the production of ahighly diverse repertoire of human antibodies, including monoclonalantibodies. This is because (1) the exogenous immunoglobulin genesretain their cis regulatory elements and are subject to the hostanimal's normal variable (V), diversity (D), and joining (J)recombinational events; (2) the exogenous immunoglobulin genes areexpressed in a similar fashion as endogenous immunoglobulin loci; and(3) the resulting antibodies apparently support normal B lymphocyticdevelopment and humoral responses.

The exogenous immunoglobulin genes may be introduced into the animals asan entire immunoglobulin locus, a part of an immunoglobulin locus, or asa “minilocus” in which a more complete exogenous Ig locus is mimickedthrough the inclusion of a handful of the individual genes from that Iglocus. Furthermore, transgenic animals may be engineered to expresstransgenes that encode modified antibodies such as single-chainantibodies or chimeric antibodies.

TWEAK agonists or antagonists useful in the methods of the invention mayalso be TWEAK polypeptides, or fragments, analogs, muteins, or mimeticsthereof, as described herein. Analogs can differ from the naturallyoccurring TWEAK amino acid sequence, or in ways that do not involve thesequence, or both. In a preferred embodiment, the TWEAK polypeptideanalogs are muteins. Methods of generating muteins are well known in theart of molecular biology, and include altering DNA molecules by randommutagenesis, site directed mutagenesis, deletions and truncations.Techniques for mutagenizing DNA are well known in the art, and includepolymerase chain reaction (PCR) mutagenesis, saturation (i.e. chemicalor radiation) mutagenesis, chemical DNA synthesis, alanine scanningmutagenesis, oligonucleotide-mediated mutagenesis (hybridization to aDNA template in vitro followed by enzymatic elongation), cassette(recombinant) mutagenesis, and combinatorial mutagenesis (introductionof random degenerate sequences into the TWEAK DNA).

The TWEAK polypeptides bind to TWEAK receptors, to other TWEAKpolypeptides, or to other TWEAK-interacting partners. The TWEAKfragments may be membrane bound, and may be delivered in pharmaceuticalcompositions that comprise liposomes or other cellular or pseudocellulardelivery systems. The TWEAK fragments may also be soluble TWEAKpolypeptides that contain either a truncation or internal deletion thatremoves the transmembrane domain. Furthermore, the TWEAK polypeptidesuseful in the methods of the invention may result in either no TWEAKresponse, or an altered TWEAK response. Examples of such TWEAKpolypeptides are analogs of the TWEAK protein, including deletion ortruncation mutants, peptides containing one or more amino acidsubstitutions, TWEAK mimetics, as well as non-amino acidsequence-modified TWEAK polypeptides.

TWEAK agonists or antagonists useful in the methods of the invention mayalso be TWEAK receptor polypeptides, or fragments, analogs, muteins, ormimetics thereof, as described herein. The TWEAK receptor polypeptidesare bound by TWEAK polypeptides, to other TWEAK receptor polypeptides,or to other TWEAK receptor-interacting partners. The TWEAK receptorfragments may be membrane bound, and may be delivered in pharmaceuticalcompositions that comprise liposomes or other cellular or pseudocellulardelivery systems. The TWEAK receptor fragments may also be soluble TWEAKreceptor polypeptides that contain either a truncation or internaldeletion that removes the transmembrane domain. Furthermore, the TWEAKreceptor polypeptides useful in the methods of the invention may resultin either no TWEAK response, or an altered TWEAK response. Examples ofsuch TWEAK receptor polypeptides are analogs of TWEAK receptor proteins,including deletion or truncation mutants, peptides containing one ormore amino acid substitutions, TWEAK receptor mimetics, as well asnon-amino acid sequence-modified TWEAK receptor polypeptides.

Moreover, TWEAK agonists or antagonists useful in the methods of theinvention may be organic or inorganic compounds. The organic compoundsmay be either small organic compounds, such as those found in chemicallibraries well known in the art. Other organic compounds include, butare not limited to, nucleic acids, peptides, saccharides, lipids andfatty acids, steroids, or derivatives thereof. Inorganic compounds maybe silica based or other minerals and salts. The organic or inorganiccompounds may bind to TWEAK polypeptides, TWEAK receptor polypeptides,or other TWEAK interacting partners, as described herein.

Non-sequence modifications of the TWEAK or TWEAK receptor polypeptidesmay result from in vivo or in vitro chemical derivatization thepolypeptides, and include, but are not limited to, changes inacetylation, methylation, phosphorylation, carboxylation, oxidationstate, or glycosylation. In addition, chemical derivatization mayinvolve coupling to organic polymers such as polyethylene glycol (PEG)or other polymers known in the medicinal arts. Therefore, a TWEAKpolypeptide analog may result from a non-amino acid sequencemodification.

The TWEAK or TWEAK polypeptides may be expressed as fusion proteins.Fusion proteins are well known in the art. A person of skill in the artmay choose from a wide variety of fusion partner moieties, includingthose from prokaryotes and eukaryotes.

According to this invention, any individual, including humans andanimals, may be treated in a pharmaceutically acceptable manner with apharmaceutically effective amount of a TWEAK agonist or antagonist orcompositions comprising such an agent, for a period of time sufficientto treat a TWEAK-related condition in the individual to whom they areadministered over some period of time. Alternatively, individuals mayreceive a prophylactically effective amount of a TWEAK agonist orantagonist, or compositions comprising such an agent, which is effectiveto prevent a TWEAK-related condition in an individual to whom they areadministered over some period of time. TWEAK agonists or antagonistsuseful in the methods of the invention may be formulated inpharmaceutical compositions by the methods disclosed herein and may bedelivered by parenteral route, injection, transmucosal, oral,inhalation, ocular, rectal, long-acting implantation, topical,sustained-released or stent-coated means. TWEAK agonists or antagonistsmay be in a variety of conventional forms employed for administration.These include, for example, solid, semi-solid and liquid dosage forms,such as liquid solutions or suspension, slurries, gels, creams, balms,emulsions, lotions, powders, sprays, foams, pastes, ointments, salves,and drops.

In addition, TWEAK agonists or antagonists may be delivered via a genetherapy route. Briefly, nucleic acid molecules encoding proteins orexpressing antisense molecules are delivered to a subject utilizing anyof the vectors known in the art to be suitable for delivering thenucleic acid molecules to the target tissues or organs. Typical vectorsinclude liposomes, plasmids, and viral vectors (e.g., retroviruses,adenoviruses and adeno-associated viruses).

The most effective mode of administration and dosage regimen of TWEAKagonists or antagonists, or compositions comprising them, will depend onthe effect desired, previous therapy, if any, the individual's healthstatus, the status of the condition itself, the response to the TWEAKagonists or antagonists and the judgment of the treating physician.TWEAK agonists or antagonists, or compositions comprising them, may beadministered in any dosage form acceptable for pharmaceuticals orveterinary preparations, at one time or over a series of treatments.

The amount of TWEAK agonists or antagonists, or compositions comprisingthem, which provides a single dosage will vary depending upon theparticular mode of administration, the specific TWEAK agonists orantagonists, or composition, dose level and dose frequency. A typicalpreparation will contain between about 0.01% and about 99%, preferablybetween about 1% and about 50%, of TWEAK agonists or antagonists orcompositions thereof (w/w).

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a TWEAK agonist or antagonist isbetween about 0.005-10.00 mg/kg body weight, more preferably betweenabout 0.05-1.0 mg/kg body weight.

TWEAK agonists or antagonists, or compositions comprising them, may beadministered alone, or as part of a pharmaceutical or veterinarypreparation, or as part of a prophylactic preparation, with or withoutadjuvant. They may be administered by parenteral or oral routes. Forexample, they may be administered by oral, pulmonary, nasal, aural,anal, dermal, ocular, intravenous, intramuscular, intraarterial,intrapeutoneal, mucosal, sublingual, subcutaneous, transdermal, topicalor intracranial routes, or into the buccal cavity. In eitherpharmaceutical or veterinary applications, TWEAK agonists or antagonistsmay be topically administered to any epithelial surface. Such surfacesinclude oral, ocular, aural, anal and nasal surfaces. Pharmaceuticalcompositions may be produced by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in a conventional manner, using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. The appropriateformulation will be dependent upon the intended route of administration.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. For ocular administration, suspensions in anappropriate saline solution are used, as is known in the art.

For oral administration, the TWEAK agonists or antagonists may beformulated readily by combining the active agents with conventionalpharmaceutically acceptable carriers. TWEAK agonists or antagonists maybe formulated as tablets, pills, liposomes, granules, spheres, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient to be treated.

TWEAK agonists or antagonists, or compositions comprising them, may alsocomprise any conventional carrier or adjuvant used in pharmaceuticals orveterinary preparations. These carriers and adjuvants include, forexample, Freund's adjuvant, ion exchanges, alumina, aluminum stearate,lecithin, buffer substances, such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, waters, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium, trisilicate, cellulose-based substances andpolyethylene glycol. Adjuvants for topical or gel base forms mayinclude, for example, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols.

Pharmaceutical compositions for oral use can be obtained as a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include fillers suchas sugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillerssuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All compositions fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, TWEAK agonists or antagonists areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator, may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

TWEAK agonists or antagonists may be formulated for either parenteraladministration by injection, e.g., by bolus injection, or continuousinfusion. The agents may be formulated in aqueous solutions, aqueoussuspensions, oily suspensions, or emulsions, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative

Typical aqueous solution formulations include physiologically compatiblebuffers such as Hanks solution, Ringer's solution, or physiologicalsaline buffer. Typical oily suspensions may include lipophilic solventsor vehicles that include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspensions may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, TWEAK agonists or antagonists may be in powder form forconstitution with a suitable vehicle, such as sterile pyrogen-freewater, before use.

The TWEAK agonists or antagonists may also be formulated in rectalcompositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

In addition to the formulations described, TWEAK agonists or antagonistsmay also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

A pharmaceutical carrier for TWEAK agonists or antagonists which arehydrophobic is a co-solvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Theco-solvent system may be the VPD co-solvent system. VPD is a solution of3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant polysorbate80, and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone; and othersugars or polysaccharides may be substituted for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are examples ofdelivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents, such as dimethylsulfoxide also may be employed, although theymay display a greater toxicity.

Additionally, TWEAK agonists or antagonists may be delivered using asustained-release system, such as semipermeable matrices of solidhydrophobic polymers containing the therapeutic agent. Varioussustained-release materials are available and well known by thoseskilled in the art. Sustained-release capsules may, depending on theirchemical nature, release the compounds for a few weeks up to over 100days.

Depending on the chemical nature and the biological stability of theTWEAK agonist or antagonist, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

TWEAK agonists or antagonists may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms.

TWEAK agonists or antagonists may also be formulated into pharmaceuticalcompositions useful for coating stents, for the treatment of theTWEAK-related heart conditions.

The present invention also relates to a method for identifying a TWEAKagonist or antagonist. Such TWEAK agonists or antagonists are useful forthe treating TWEAK-related conditions, i.e., diseases, settings ofinjury or other pathological conditions of tissues wherein a receptorfor TWEAK, e.g. FN14, is expressed. Those conditions include fibrosisand diseases of the heart (e.g. cardiomyopathies), kidney, lung, liver,skin, skeletal muscle, lipid metabolism (e.g. obesity), gastrointestinaltract, pancreas, reproductive organs, neural tissue (includingneurodegeneration), cartilage, bone and connective tissue. Such TWEAKagonists or antagonists are also useful for promoting tissue replacementby modulating the behavior of progenitor cells in vivo or in vitroaccording to the present invention.

One embodiment of the method for identifying a TWEAK antagonistcomprises the steps of: 1) exposing a transgenic test animal thatexpresses an exogenous DNA encoding a TWEAK polypeptide, or a fragment,analog, mutein, or mimetic thereof, to a compound which is a candidateTWEAK antagonist; 2) comparing the fibrotic, cardiac, kidney, liver,lung, skin, skeletal muscle, lipid, gastrointestinal tract, pancreas,reproductive organs, neural, cartilage, bone or connective tissue fromthe transgenic test animal to the same organ or tissue from a referenceanimal that expresses the exogenous DNA but was not exposed to thecompound; and 3) determining whether the compound has affected thefibrotic, cardiac, kidney, liver, lung, skin, skeletal muscle, lipid,gastrointestinal tract, pancreas, reproductive organs, neural,cartilage, bone or connective tissue. In another embodiment, thetransgenic test animal is either a mammal or a non-mammal, as disclosedherein.

The transgenic animals disclosed herein express exogenous DNAs encodingTWEAK polypeptides, wherein the expression results in a TWEAK-relatedcondition. In the examples, transgenic mice were generated that expressexogenous TWEAK proteins in either a truncated, soluble form or in afull-length, membrane-bound form. The mice that express the exogenousTWEAK proteins revealed phenotypes that include non-inflammatory dilatedcardiomyopathy, congestive heart failure, liver epithelial cellhyperplasia, hepatocyte vacuolation, liver injury and inflammatorykidney conditions, such as multifocal inflammation, non-inflammatorykidney conditions, such as tubular nephropathy, cysts, glomerularnephropathy, kidney tubular hyperplasia, kidney fibrosis andinflammatory lung conditions. Furthermore, wild-type mice that wereinfected with viral vectors that express exogenous TWEAK proteins showedductal hyperplasia, hepatocyte death, liver fibrosis and liver injury aswell.

Having these animals in hand, persons of skill in the art have apowerful method for drug discovery. Specifically, the animals thatexpress exogenous TWEAK proteins represent a model system for practicinga method for the discovery of TWEAK agonists or antagonists useful forthe prevention or treatment of the TWEAK-related conditions disclosedherein.

In preferred embodiments, the animals useful in these model systems areeither mammalian or non-mammalian. In more preferred embodiments, themammalian animals are mice, rats, hamsters, rabbits, dogs, cats, cows,pigs, goats, horses, sheep, guinea pigs and primates. In other morepreferred embodiments, the non-mammalian animals are birds, fish,amphibians, insects, and invertebrates.

The exogenous DNA encoding the TWEAK polypeptide is expressed in thetransgenic animals via expression control sequences that control theexpression of the exogenous DNA in the animal. Expression controlsequences that control transcription include, e.g., promoters, enhancerstranscription termination sites, locus control regions, RNA polymeraseprocessivity signals, and chromatin remodeling elements. Expressioncontrol sequences that control post-transcriptional events includesplice donor and acceptor sites and sequences that modify the half-lifeof the transcribed RNA, e.g., sequences that direct poly(A) addition orbinding sites for RNA-binding proteins. Expression control sequencesthat control translation include ribosome binding sites, sequences whichdirect targeted expression of the polypeptide to or within particularcellular compartments, and sequences in the 5′ and 3′ untranslatedregions that modify the rate or efficiency of translation.

Preferred expression control sequences for TWEAK expression in thetransgenic animals include viral elements that direct high levels ofprotein expression, such as promoters and/or enhancers derived fromretroviral LTRs, cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. In one embodiment, the DNA encodinga TWEAK polypeptide is driven by the alpha1 anti-trypsin (AAT) promoter.For further descriptions of viral expression control elements, andsequences thereof, see, e.g., U.S. Pat. Nos. 5,168,062; 4,510,245; and4,968,615.

The exogenous DNA may also be expressed in the transgenic animals fromtissue-specific expression control elements, including promoters.Tissue-specific expression control elements are known in the art.Non-limiting examples of suitable tissue-specific promoters include theliver-specific albumin promoter (Pinkert et al., Genes Dev. 1:268-277(1987)), lymphoid-specific promoters (e.g., Calame and Eaton, Adv.Immunol. 43:235-275 (1988); Winoto and Baltimore, EMBO J. 8:729-733(1989); Banerji et al., Cell 33:729-740 (1983); and Queen and Baltimore,Cell 33:741-748 (1983)), neuron-specific promoters (e.g., Byrne andRuddle Proc. Natl. Acad. Sci. USA 86:5473-5477 (1989)),pancreas-specific promoters (e.g., Edlund et al., Science 230:912-916(1985)), mammary gland-specific promoters (e.g., U.S. Pat. No. 4,873,316and European patent application 264,166), and developmentally-regulatedpromoters (e.g., Kessel and Gruss, Science 249:374-379 (1990); Campesand Tilghman, Genes Dev. 3:537-546 (1989)). Other non-limiting examplesof tissue-specific promoters include the cardiac tissue promoter alphamyosin heavy chain promoter (α MHC), the skin tissue promoter keratin-14(K14), the lung tissue promoter surfactant protein C (SPC), and thekidney tissue promoters Ksp-cadherin and kidney androgen-regulatedprotein (KAP). The exogenous DNA may also be expressed from an inducibleeukaryotic promoter, such as the metallothionine (MT) promoter, or otherinducible eukaryotic promoters known in the art.

In one embodiment of the present invention, the TWEAK polypeptideexpressed in the transgenic animals of the invention may be afull-length TWEAK polypeptide. In another embodiment, the polypeptidesexpressed in the transgenic animals are fragments of the TWEAKpolypeptide. In a preferred embodiment, the TWEAK polypeptide fragmentsare soluble TWEAK polypeptides.

In another embodiment, the invention relates to transgenic animals thatexpress an exogenous DNA encoding a TWEAK polypeptide in a tissueselected from the group consisting of: heart; blood; vessel; lungs;liver; kidney; brain; placenta; skeletal muscle; pancreas; spleen;lymph; thymus; appendix; peripheral blood lymphocyte; gastrointestinaltract; neurons; skin; adipocyte; cartilage; bone; connective tissue. Inone embodiment, the TWEAK DNA is expressed from a constitutive promoter.In another embodiment, the DNA is expressed from an inducible promoter.In another embodiment, the DNA is expressed from a tissue specificpromoter.

The present invention also relates to methods of identifying TWEAKagonist compounds that may act as therapeutic agents for treatment ofTWEAK-related conditions or for promoting tissue replacement bymodulating the behavior of progenitor cells in vivo or in vitroaccording to the present invention. Agonist candidate compounds may beadministered to normal animals and their effect on organ systemsassessed. Fibrotic, cardiac, liver, kidney, lung, skin, skeletal muscle,lipid, gastrointestinal tract, pancreas, reproductive organs, neural,cartilage, bone or connective tissue from the treated animal is thencompared to the same tissue from an untreated animal; it is therebydetermined whether the compound has induced a biological effect in anyof said tissues.

This invention also relates to methods of identifying TWEAK regulatedgenes that may act as therapeutic targets for treatment of TWEAK-relatedconditions. For example, RNA profiling could be carried out in TWEAKtransgenic animals as compared to normal animals in various tissues anddrug targets thus identified.

It is a further objective of the invention to provide methods ofaffecting progenitor stem cell proliferation or differentiation,including that of mesenchymal stem cell types that give rise to musclecells, cartilage, bone or connective tissue cell types such as stromalcells, fibroblasts, adipocytes and dermal cells. It is also an objectiveof the invention to provide methods of affecting the proliferative ordifferentiative ability of oval cells, which can give rise to biliaryepithelial cells or hepatocytes and kidney progenitors, which can giverise to tubular epithelium.

EXAMPLES

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any manner.

Example 1 Generation of TWEAK Transgenic Mice

In order to identify target organ(s) for TWEAK activity and thebiological consequences of TWEAK signaling in vivo, two murine TWEAKexpression constructs were created and used for the overexpression ofTWEAK peptides in normal (C57Bl/6×DBA/2)F1 and (C57Bl/6×SJL/J)F2 miceusing standard transgenic techniques. R. S. Williams and P. D. Wagner,J. Applied Physiology 88:1119-1126 (2000). The TWEAK expressionconstructs used were as follows: (1) A TWEAK cDNA from amino acids101-249 of SEQ ID NO:1, encoding a soluble form of murine TWEAK(designated sTWEAK) downstream of a murine IgG signal sequence wasinserted into the CH269 expression vector (a derivative of vector PCDEP4(Invitrogen) containing the SV40 poly A addition sequence) downstream ofthe human alpha1 anti-trypsin (AAT) promoter and a beta-globin intronand upstream of the human growth hormone (hGH) poly A sequence; and (2)A cDNA encoding the full-length, transmembrane form of the protein(designated FL-TWEAK) corresponding to amino acids 1-249 of SEQ ID NO:1was inserted into the pBlueScript expression vector (as described inDesplat-Jego et al., J. Neuroimmunology 133:116-123 (2002)) containingthe SV40 poly A addition site. The FL-TWEAK sequence plus the poly Aaddition site was then isolated and cloned into another vector; afragment containing the ApoE enhancer-human AAT promoter regulatoryregion was then inserted upstream to create the expression vector CA300.The AAT promoter has been shown to direct transcription primarily inliver and at lower levels in other tissues including kidney. P. Koopmanet al., Genes Dev. 3:16-25 (1989).

For the sTWEAK transgene construct, 23 independent transgenic founderswere identified by tail DNA PCR using probes corresponding to thesequence of nucleotides 468 to 488 (5′ primer) and the complementarystrand sequence for nucleotides 693 to 713 (3′ primer) of SEQ ID NO:2.In addition, a serum ELISA for TWEAK revealed that 10 of these 23founder animals had detectable levels of TWEAK in their serum, rangingfrom 0.06-3.0 micrograms/ml. The remaining 13 founders had no detectableserum TWEAK, i.e. <10 ng/ml. Nine of the 10 PCR+, serum TWEAK+ foundersbegan to exhibit ill health at approximately 4-5 months of age. Weightloss, hunched posture, unkempt fur and bulging eyes were noted. Five ofthese founders died unexpectedly and therefore the remaining four thatshowed signs of illness were sacrificed with signs of ill health. Incontrast, only 1 of 13 PCR+ serum negative founders exhibited ill healthor died. Also, 0 of 4 PCR negative littermates exhibited illhealth/died.

For the FL-TWEAK transgene construct, two independent transgenicfounders were identified by tail DNA PCR and Northern blot analysis forTWEAK mRNA expression in liver tissue. In addition, a serum ELISA forTWEAK revealed that neither of these two founder animals had detectablelevels of TWEAK in their serum, i.e., less than 10 nanograms/ml. TheFL-TWEAK Tg founder mice did not exhibit a clinically observablephenotype.

Example 2 Overexpression of TWEAK in Mice Infected with an AdenoviralVector Delivering an Exogenous DNA Encoding sTWEAK

In order to identify the biological effects of overexpression of TWEAKin vivo, 8-10 week old C57BL/6 female adult mice were infected with areplication-defective adenoviral vector with a cytomegalovirus (CMV)promoter driving the cDNA for either murine sTWEAK (“Adeno-TWEAK”) orjellyfish green fluorescent protein (“GFP”) using standard adenoviraltechniques as described in Tao et al., Molecular Therapy 3:28-35 (2001).An adenoviral vector comprising GFP (“Adeno-GFP”) was used as thenegative control. To determine whether mice were successfully infectedwith the Adeno-TWEAK construct, TWEAK protein levels in the serum weredetermined and monitored at various time points using standard ELISAassays.

Systemic overexpression of murine sTWEAK in the adult mice inducedtissue changes in at least three major organs: liver, kidney and heart.See, Table 1. The phenotypes of these adenoviral construct-expressingmice were compared with the phenotypes of TWEAK Tg mice from Example 1in Table 1. These observations are discussed in more detail in thefollowing examples below.

TABLE 1 TWEAK Overexpression Induces Tissue Remodeling in Adult MiceAdeno- Adeno-TWEAK GFP TWEAK in Adult in Adult ORGAN PHENOTYPE Tg MiceMice Liver Bile Duct + + − Hyperplasia Hepatocyte − + − Death KidneyTubular + + − Hyperplasia Heart Dilated + + − Cardiomyopathy

Example 3 sTWEAK and FL-TWEAK Induce Dilated Cardiomyopathy

Four of the surviving PCR+ serum sTWEAK+ founders from Example 1 weresacrificed and examined for gross morphological abnormalities. See,Table 2. Macroscopic observation at the time of necropsy revealedenlarged hearts, some 2-3 fold increased in size as compared to those ofnormal animals. Since the enlarged heart phenotype was observed inmultiple independent sTWEAK transgenic founders, it is highly unlikelyto be due to independent insertional events. Furthermore, an analysis ofthe serum chemistry of the sTWEAK transgenic mice showed elevatedcardiac specific creatine kinase.

TABLE 2 sTWEAK Transgenic Mice FOUNDERS SERUM TWEAK PHENOTYPE 10 PCR+0.06-3.0 μg/ml 9/10 dead at 4-5 months 4/5 submitted forhistophathological examination had enlarged heart 13 PCR+ <10 ng/ml 1/13dead at 6 months with enlarged heart  4 PCR− — 0/4 dead at 6 months

The enlarged heart phenotype was also observed in individual mice fromone FL-TWEAK transgenic line that was established through successivebackcross onto the C57BL/6 strain. See, Table 3. The FL-TWEAK transgenenegative littermates showed no heart abnormalities.

TABLE 3 FL-TWEAK Transgenic Mice INDIVIDUALS SERUM TWEAK PHENOTYPE 7PCR+ — 7/7 showed enlarged hearts at 5 months 4 PCR− <10 ng/ml 0/4 deadat 5 months

Taken together, these data strongly indicate that the enlarged heartphenotype is TWEAK-dependent.

Histopathological analysis of the hearts from the sTWEAK transgenic andFL-TWEAK transgenic mice showed similar findings. Low power microscopyof a FL-TWEAK transgenic heart (“Tg”) as compared to a normal heart froma transgene negative (“NTg”) littermate is shown in FIG. 1. The FL-TWEAKtransgenic heart shown is also representative of TWEAK transgenic heartsfrom sTWEAK transgenic mice (PCR+, serum TWEAK+). The transgenic heartsexhibited dilated cardiomyopathy, characterized by dilation of theventricles and atria. Consistent with this defect, atrial andventricular thrombosis in many of the animals was noted (FIG. 1).Analysis of lung and liver tissue revealed congestion of the bloodvessels in some animals.

Higher power microscopy revealed other histopathological findings in theheart, including myocardiocyte hypertrophy and karyomegaly. Notably, thehistopathological analysis of the ventricles in TWEAK transgenic miceshowed no signs of inflammation. Therefore, the observed TWEAK-relatedcardiomyopathy is non-inflammatory in nature.

Serum chemistry analysis on terminal bleeds from sTWEAK transgenic mice(3 founders and 1 progeny mouse) showed abnormally high levels ofcreatine kinase (CK) specifically in the heart (i.e. the MB type of CK),confirming a significant level of cardiac stress/injury.

C57BL/6 female mice of 8-10 weeks of age infected with Adeno-TWEAK (see,Example 2) showed dilated cardiomyopathy which was apparent three weekspost infection as compared to mice infected with the negative controlAdeno-GFP virus. In TWEAK-infected mice, the hearts were characterizedby dilated chambers, as shown by histopathology (FIG. 2).

Taken together, TWEAK was shown to play a critical role incardiomyopathies, including dilated cardiomyopathy, and congestive heartfailure (CHF).

Example 4 TWEAK Causes Liver Epithelial Cell Hyperplasia, HepatocyteVacuolation, Hepatocellular Death, Bile Duct Hyperplasia, Liver Fibrosisand Liver Injury

A role for TWEAK in liver epithelial hyperplasia and hepatocytevacuolation was revealed in sTWEAK and FL-TWEAK transgenic mice as wellas injury in wild-type mice infected with an adenovirus harboring a DNAthat expresses a sTWEAK polypeptide.

The livers of TWEAK Tg mice from Example 1 showed substantial biliaryduct and oval cell hyperplasia by 2 weeks of age as compared to NTgmice. See, FIG. 3. As shown in Table 4, even at serum TWEAK levels of<10 ng/ml, the livers of two FL-TWEAK transgenic mouse founders showedmild biliary duct and oval cell hyperplasia. FL-TWEAK transgenic mousebackcrosses into the C57BL/6 background revealed substantial biliaryduct and oval cell hyperplasia (Table 4).

TABLE 4 FL-TWEAK Transgenic Mice MICE SERUM TWEAK PHENOTYPE 2 Founders<10 ng/ml Mild biliary duct and oval cell hyperplasia 1 Founder —Prominent biliary duct backcrossed and oval cell hyperplasia intoC57BL/6

Similarly, the sTWEAK transgenic founders that have TWEAK serum levelsbetween 0.2 and 3.0 μg/ml showed considerable biliary duct and oval cellhyperplasia (Table 5).

TABLE 5 sTWEAK Transgenic Mice MICE SERUM TWEAK PHENOTYPE 9 Founders0.2-3.0 μg/ml Prominent biliary duct and oval cell hyperplasia and ovalcell hyperplasia 1 Founder   0.06 μg/ml Mild biliary duct and oval cellhyperplasia

This biliary and oval duct hyperplasia was confirmed byimmunohistochemical (IHC) staining of FL-TWEAK Tg liver sections takenfrom the Tg mice of Example 1 with the A6 mAb, which distinguishesbiliary epithelial cells and oval cells from hepatocytes (Engelhardt etal., Differentiation 45:29-37 (1990)). FIG. 4 shows an increase in A6positive cells that are associated with portal regions as well as extendout into the liver parenchyma in FL-TWEAK Tg as compared to NTg mice.Higher magnification of a hematoxylin and eosin stained section from aFL-TWEAK Tg mouse also clearly shows a marked increase in the presenceof oval cells adjacent to the biliary ducts in the portal region (FIG.5). Immunohistochemistry for the proliferating cell nuclear antigen(PCNA) confirmed an increased frequency of proliferating biliary andoval cells in, TWEAK Tg mice as compared to NTg mice as early as 2 weeksof age. At later time points, an increase in frequency of proliferatinghepatocytes in TWEAK Tg mice as compared to NTg mice was observed, i.e.,between 8 weeks and 7 months of age (not shown). Furthermore, bothFL-TWEAK and sTWEAK induced hepatocellular vacuolization in 7 month oldTg mice from Example 1 as compared to NTg littermates (FIG. 6).

C57BL/6 and BALB/c SCID mice of 8-10 weeks of age overexpressing sTWEAKusing the Adeno-TWEAK virus as in Example 2 showed substantial serumTWEAK levels. See, FIG. 7, which displays the effect of deliveringdifferent doses of adenovirus on the serum TWEAK levels measured. Micewere infected with either 10¹¹ particles of adenovirus per mouseintravenously (represented by the “B” line), 10¹⁰ particles ofadenovirus per mouse intravenously (represented by the “J” line) or 10¹¹particles of adenovirus per mouse intramuscularly (represented by the“H” line). The Adeno-sTWEAK-infected mice showed liver injury, withserum jaundice observed on days 3 and 7 in the C57BL/6 mouse backgroundand on days 3 and 4 of the BALB/c SCID mouse background. Some of theBALB/c SCID mice died on day 4.

Furthermore, the Adeno-sTWEAK-infected C57BL/6 mice as described inExample 2 also developed hepatocellular death which appeared as early as2-3 days post administration, as demonstrated by the high level of theaspartate aminotransferase (“AST”) and alanine aminotransferase (“ALT”)liver enzyme markers in TWEAK-infected livers (Adeno-sTWEAK) comparedwith control GFP-infected livers (Adeno-GFP) by day 3 (see, Table 6 andFIG. 8). By day 7 post infection, both liver enzymes also rose in theAdeno-GFP treated mice, as would be expected due to the inflammationinduced by the adenovirus vector alone. Hepatocyte death was alsoapparent in TWEAK-infected livers, as shown by the histologic morphologycharacterized by rounded up hepatocyes and shrunken, pyknotic, intenselyeosinophilic “Councilman bodies” containing fragmented nuclei (FIG. 8).Adeno-sTWEAK treated mice further developed a strong hyperplastic ductalresponse, which peaked on day 7 post infection and was still readilyapparent on day 11 (FIG. 8). In the TWEAK-infected livers, hyperplasticstructures were observed that expressed the A6 marker specific forbiliary epithelium and oval cells, as identified by bright fieldmicroscopy.

TABLE 6 Liver Enzyme Values in Ad-TWEAK And Ad-GFP Animals Adeno-GFPAdeno-sTWEAK DAY AST (U/L) ALT (U/L) AST (U/L) ALT (U/L) 3 148 110 1715672 7 2140 1545 1910 1194 11 2540 2304 795 508 20 683 420 451 320

Fn14, shown to be a cellular TWEAK receptor, was induced after exposureto liver toxins, such as galactosamine (GalN) and carbon tetrachloride(CCl₄). FIG. 9 shows that Fn14 is not detectable in normal adult mouseliver as measured by in situ hybridization (ISH) using a radiolabeledprobe for Fn14 and dark field microscopy. However, Fn14 is highlyinduced following CCl₄ injury. Similar results were obtained after GalNinjury (not shown).

Adeno-TWEAK-infected C57BL/6 mice as described in Example 2 alsorevealed upregulation of Fn14 in the hepatocytes and some hyperplasticstructures, as observed in Adeno-sTWEAK livers compared with Adeno-GFPcontrol livers (data not shown).

The role of Fn14 was further demonstrated in a bilary duct model whereinhepatic injury in 10 week old C57BL/6 mice was induced by ligation ofthe biliary duct as described by Liu et al., Hepatology 28:1260-1268(1999); Olynyc et al., Am. J. Pathol. 152:347-352 (1998). The commonbile duct was ligated on day 0 by surgery and five C57BL/6 mice of 10weeks of age were then euthanized on day 4 and day 8 post surgery.Paraffin sections of liver were then prepared and the expression ofTWEAK and Fn14 were determined by in situ hybridization using aradiolabeled murine TWEAK and FN14 anti-sense probe encompassing thecomplete FN14 gene. As shown in FIG. 10, by day 4, Fn14 was expressedstrongly in biliary epithelial cells in bile ducts but not inhepatocytes. By day 8, Fn14 expression in biliary epithelial cellsdecreased significantly but was still detectable at low levels in somemice (data not shown). However, TWEAK expression did not change and wasnot detectable in this bile duct ligation model. These results show thatFn14 expression is upregulated in biliary epithelial cells in responseto certain liver injuries and, thus, plays an important role in liverfibrosis.

Taken together, these observations show that TWEAK is an importantfactor in liver epithelial cell hyperplasia, hepatocyte vacuolation,liver injury, heptocellular death, bile duct hyperplasia and liverfibrosis.

Example 5 FL-TWEAK and sTWEAK Cause Kidney Disease

FL-TWEAK transgenic mice from Example 1 showed marked kidney disease,including mild multifocal inflammation, tubular nephropathy, cysts,glomerular nephropathy, tubular basophilia, tubular dilatation, tubularvacuolation and hyaline casts.

Adeno-TWEAK-infected C57BL/6 mice of 10 weeks of age as described inExample 2 revealed glomerular nephropathy and tubular hyperplasia ascompared to negative control Adeno-GFP-infected mice. Also, a role forTWEAK in Alports syndrome was shown by increased Fn14 expression in amouse model of Alports disease. Furthermore, a role for TWEAK in kidneyfibrosis was demonstrated in the murine model of unilateral ureteralobstruction-induced kidney fibrosis by treatment with a TWEAKantagonist.

Expansion of the cortical interstitium is typically due to edema orinfiltration with acute or chronic inflammatory cells and fibroustissue. FL-TWEAK transgenic mice from Example 1 showed tubularnephropathy and mild, multifocal interstitial inflammation. Morespecifically, kidney cross-sections comparing a non-transgenic mousewith FL-TWEAK transgenic mice showed pronounced tubular basophilia at 8weeks of age (FIG. 11, middle panel).

Glomerular nephropathy may be characterized by an infiltration ofleukocytes, both neutrophils and monocytes, and proliferation ofendothelial, epithelial and mesangial cells. FL-TWEAK transgenic mice asdescribed in Example 1 showed marked glomerular nephropathy as evidencedby hypercellularity of the mesangial cells and hypertrophy of capsularepithelia and mild capsular thickening with basophilia of adjacenttubular epithelium (FIG. 12). Also, FL-TWEAK transgenic mice showeddilation of the urinary space leading to formation of glomerular cystswith mild peri-glomerular fibrosis (FIG. 11, lower right panel), ascompared to normal murine glomerular morphology (FIG. 11, upper rightpanel).

The tubular basophilia observed in FL-TWEAK Tg mice is indicative ofincreased RNA in the cytoplasm of these tubular cells, i.e.transcriptional activity, and suggested that these were proliferatingcells. Proliferating Cell Nuclear Antigen (PCNA) staining confirmed thatthere was a subset of tubular cells proliferating in the kidneys ofTWEAK-Tg mice as described in Example 1 and that these corresponded tothe basophilic tubules (FIG. 13). In order to determine whether thebasophilic tubules were proximal or distal tubules, three serial tissuesections from TWEAK Tg mice were stained (1) with hemotoxylin and eosin(H&E) to localize the basophilic tubules, (2) with a lectin specific forproximal tubules (lectin from T. Purpureas) and (3) with a lectinspecific for distal tubules. FIG. 14 shows that the basophilic(proliferating) tubules in TWEAK Tg mice as described in Example 1 donot express either the proximal or distal tubular epithelial marker.

The presence of proliferating tubules that lack at least some epithelialmarkers in the TWEAK Tg mice is consistent with a model for settings ofkidney injury where cells derived from the S3 segment of the proximaltubule exhibit the properties of progenitor cells, i.e. they begin toproliferate and express mesenchymal cell markers indicative ofdedifferentiation. Subsequent differentiation of these cells may play arole in tissue repair through the regeneration of new tubules (Witzgallet al., J. Clin. Invest. 93:2175-2188 (1994)). Alternatively, there maybe proliferation and differentiation of a pre-existing progenitorpopulation that resides in the S3 region.

The presence of proliferating cells that lack some epithelial markers inTWEAK Tg mice is also consistent with a model for kidney development,wherein epithelial tubules are formed from mesenchymal progenitors thatundergo differentiation, thereby acquiring epithelial markers andproperties characteristic of tubules.

Similarly, infection of 10 week old C57BL/6 mice with an Adeno-sTWEAKvirus, as described in Example 2, induced glomerular nephropathy andbasophilia of the tubular epithelium as well as occasional thickeningand hyperplasia of the glomerular capsula by day 11 post infection (FIG.15). This was in contrast to the normal histology observed in thenegative control Adeno-GFP-infected mice. Furthermore, the basophilia,which is indicative of epithelial cell proliferation, was apparent byday 3 but peaked around one week post administration.

Consistent with a role for TWEAK in kidney disease, TWEAK mRNA was shownto be expressed widely in adult C57BL/6 mouse kidney (FIG. 16), and Fn14mRNA was shown to be expressed in the proximal tubules of the innercortex/outer medulla (FIG. 17), as shown by in situ hybridization (ISH)using radiolabeled TWEAK and Fn14 antisense probes and revealed by darkfield microscopy. Also, Fn14 mRNA was shown to be induced in the kidneysof mouse models for Alport syndrome. This is shown in FIG. 18 as thefold increase in Fn14 mRNA in two individual Alport mice relative towildtype animals as disease progresses in the Alport mice from 4 to 7weeks of age.

The role of TWEAK in a murine model of Alport disease was directlytested by treatment with a TWEAK antagonist, a murine Fn14-Fc fusionprotein. Two groups of 5 Alport knockout (KO) mice prepared according toCosgrove et al., Genes Dev. 10(23):2981-2992 (1996), were treated withcontrol IgG2a (muP1.17), or muFN14-Fc fusion protein (prepared by Biogen(Cambridge)). The control IgG2a used is the murine myeloma protein P1.17produced from a hybridoma and purified by standard mAb purificationprocedure. The muFN14-Fc is a fusion protein of the extracellular domainof murine Fn14 and the Fc region of murine IgG2a. The fusion protein wasproduced either in human 293 embryonic kidney cells or in Chinesehamster ovary (CHO) cells and purified by standard mAb purificationprocedures. The first treatment was at the age of three weeks with adose of one hundred microgram of protein by an intraperitoneal (IP)route. Treatments continued for the next four weeks with the same doseadministered twice a week. Mice were sacrificed at the end of the 7thweek (7 week old). Kidneys were collected and embedded in paraffin andfrozen. The extent of kidney fibrosis and inflammation was scored byglomerular morphology from H&E staining of paraffin sections, activatedmyofibroblast with smooth muscle actin staining, and activated monocytesby CD11b staining of frozen sections. Smooth muscle actin and CD11bstained sections were used to quantitate positively stained areas toassess extent of fibrosis and inflammation, respectively, by theMetaMorph computer program. Results of analysis show health of glomeruliin FN14-Fc treated mice was greatly improved (59% glomeruli withpathology in control Ig treated as compared to only 39% with pathologyin Fn14-Fc treated, P value=0.03). Glomerular pathology is characterizedby presence of crescents and/or glomerular fibrosis. In addition,fibrosis in the cortical area of the kidney in treated mice wassignificantly reduced as measured by alpha smooth muscle actin staining,p value=0.04. There was also a general trend in reduction of monocyteinfiltration in FN14-Fc treated mice. These results clearly indicatethat FN14-Fc treatment of Alports mice reduces fibrosis in the corticalarea of the kidney and improves the general morphology of glomeruli.

The role of TWEAK was also tested in the murine model of unilateralureteral obstruction-induced kidney fibrosis by treatment with a TWEAKantagonist, a hamster anti-TWEAK monoclonal antibody. In the mouse modelfor renal fibrotic progression, a ureter is ligated, resulting inunilateral ureteral obstruction (WO). (Klahr et al., Am J Kidney Dis18:689-699 (1991); Moriyama et al., Kidney Int 54(1):110-119 (1998). WOcauses progressive nephrosclerosis without near-term renal failure inmice because the unobstructed kidney can maintain relatively normalrenal function. While the obstructed kidney undergoes rapid globalfibrosis, the unobstructed kidney undergoes adaptive hypertrophy.

The impact of TWEAK antagonist treatment on UUO-induced renal fibrosiswas quantitated morphometrically. Four groups of eight viralantigen-free C57BL/6 male mice (Jackson Laboratories, Bar Harbor Me.),8-10 weeks of age were used. The mice were divided into the followinggroups: PBS alone (VEH), control hamster anti-Keyhole Limpet Hemocyanin(KLH) antibody (HA4/8; purchased from BD Biosciences (San Jose)),hamster anti-mouse TWEAK antibody (AB.G11; prepared by Biogen(Cambridge)), soluble murine TGF-β receptor Ig (TGF-βR, positivecontrol; prepared by Biogen (Cambridge)) and unoperated (UNOP).

To induce kidney fibrosis, the left ureter was aseptically isolated andtied off in the kidney of the operated side on day 0 as described inHammad et al., Kidney Int 58:242-250 (2000). The following groups: PBS,HA4/8 and AB.G11 (anti-TWEAK mAb) were additionally treated on days 2,6, and 9 post surgery and the sTGF-βR-Ig group on days 1, 3, 6 and 8. Onday 10 post surgery, the left ligated kidney was removed and halvedtransversely through the center of the renal pelvis and prepared forparaffin sectioning.

Paraffin-treated kidney sections were stained with Trichrome-Massonstaining specific for collagen. Using a Metamorph program, blue-stainingareas in Trichrome-Masson slides were measured to quantitate collagencontent in order to assess the extent of fibrosis in the operatedkidneys (FIG. 19).

Surprisingly, kidney sections from anti-TWEAK monoclonal (AB.G11)antibody-treated animals demonstrated a 42% decrease in collagen contentcompared with PBS-treated animals and a 30% decrease in collagen contentcompared with control (HA4/8) antibody-treated animals. In contrast, thekidneys from soluble TGF-β receptor Ig-treated (TGF-βR) animalsdisplayed only a 33% decrease in collagen content compared withPBS-treated animals and a 19% decrease in collagen content compared withcontrol (HA4/8) antibody-treated animals. These results clearly showthat treatment with a TWEAK antagonist, such as an anti-TWEAK monoclonalantibody, significantly reduced kidney fibrosis to a greater extent thanthat shown by soluble TGF-β receptor Ig (TGF-βR).

Taken together, the results presented herein show that TWEAK plays animportant role in inflammatory kidney conditions, such as multifocalinflammation, and in non-inflammatory kidney conditions, such as tubularnephropathy, cysts, glomerular nephropathy, Alports syndrome, tubularbasophilia, tubular dilatation, tubular vacuolation, hyaline casts,tubular hyperplasia and kidney fibrosis.

Example 6 TWEAK Causes Lung Inflammation

In cross sections of lungs from FL-TWEAK transgenic and control mice asdescribed in Example 1, marked granulomatous and lymphohistiocyticinflammation was shown in both FL-TWEAK and sTWEAK Tg mice (FIG. 20).Also, endogenous TWEAK expression was revealed in lung cells lining thebronchioles and alveoli of normal mice, as shown by in situhybridization (ISH) using radio-labeled TWEAK antisense probes andrevealed by dark field (ISH) microscopy (FIG. 21).

Consistent with a role for TWEAK in lung disease, Fn14 mRNA was shown tobe expressed widely in adult C57BL/6 mouse lung (FIG. 22) by ISH usingradio-labeled Fn14 antisense probes and revealed by dark fieldmicroscopy.

Taken together, these data show that TWEAK is an important factor inmediating inflammatory lung conditions, including granulomatous andlymphohistiocytic inflammation.

Example 7 TWEAK Inhibits Both Adipogenesis and Myogenesis

The effect of TWEAK on cellular differentiation was investigated usingtwo in vitro models of adipogenesis and myogenesis well-known in theart. (Green and Meuth, Cell 3:127-133 (1974); Yaffe and Saxel, Nature270: 725-727 (1977)).

For adipogenesis, 3T3-L1 cells were first grown to confluency in aDulbecco's Modified Eagles Media (DMEM)-based growth media and theninduced to undergo adipogenesis according to methods known in the art.Green and Kehinde, Cell 5:19-27 (1976). Briefly, cells were stimulatedon day 0 with the DMEM-based MDI media that contained dexamethasone,insulin and IBMX for two days followed by insulin-only DMEM media foranother two days. On day 5, cells were cultured in the regularDMEM-based growth media and adipogenesis was assessed on day 7 byOil-Red staining.

For myogenesis, C2C12 cells were grown to near confluency in aDMEM-based growth media and on day 0, switched to a low-serumdifferentiation media that contained 2% horse serum to triggerdifferentiation (Yaffe and Saxel, Nature 270: 725-727 (1977)). Myotubeformation was examined using a phase-contrast microscope and pictureswere taken on day 6 of differentiation.

To examine the effect of TWEAK on these two differentiation pathways,various versions of recombinant human TWEAK (TWEAK-FLAG, TWEAK orFc-TWEAK) were added on day 0 at a final concentration of 100 ng/ml andreplenished daily. TWEAK inhibited both adipogenesis and myogenesis inboth systems (FIGS. 23 and 24). The specificity of TWEAK'S inhibitoryeffect was confirmed using either the hamster anti-TWEAK monoclonalantibody AB.G11 or hFn14-Fc as the neutralizing reagent.

These results show that TWEAK plays an important role in cellulardifferentiation. The present invention therefore provides methods foraffecting cellular differentiation of the progenitor cells disclosedherein using the TWEAK polypeptides, peptides, agonists, or antagonistsdisclosed herein.

Example 8 TWEAK Binds to Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) (Cambrex Corp., East Rutherford,N.J.) were cultured in MSCGM media (Cambrex) and harvested by incubatingthem with PBS containing 5 mM EDTA, and prepared for fluorescenceactivated cell sorting (FACS) analysis.

The cells were incubated in FACS buffer containing PBS and 1% FBS alongwith 100 ng/mL of Fc-TWEAK for 1 hour on ice. After washing twice withFACS buffer, the cells were then incubated with phycoerythrin-conjugatedgoat anti-human Fc or goat anti-mouse Fc secondary antibodies at adilution of 1:200 (Jackson ImmunoResearch, West Grove, Pa.) (FIG. 25).The background staining was measured by secondary antibody stainingalone, as shown by the broken line.

As shown in FIG. 25, TWEAK binds to human mesenchymal cells, asdemonstrated by the shift in the staining profile of Fc-TWEAK comparedwith secondary antibody alone. Thus, the ability of TWEAK to bind tomesenchymal cells (a progenitor cell type capable of differentiatinginto muscle cells as well as cartilage, bone, connective tissue celltypes such as stromal cells, fibroblasts, adipocytes and dermal cells)shows that TWEAK plays an important role in the differentiation of thesecell types both in normal and disease models.

Example 9 Fn14 is Expressed on Neural Stem Cells

The expression of Fn14 was examined in the brains from embryonic day13.5 mice on a mixture of both C57BL/6 and 129/Sve background. Thebrains were subjected to in situ hybridization with the Fn14 anti-senseprobe. A positive signal was detectable in the subventricular zone ofthe embryonic ventricles, correlating with the position of neural stemcells (data not shown). These results show that Fn14 plays an importantrole in neural cellular differentiation.

Example 10 Methods for Identifying Therapeutic Agents for TreatingTWEAK-Related Conditions

In order to identify TWEAK antagonist compounds that act as therapeuticagents for the treatment of TWEAK-related conditions according to thepresent invention, a test animal, such as a mouse, is obtained thatexpresses an exogenous DNA encoding a TWEAK polypeptide, or a fragment,analog, mutein, or mimetic thereof. The animal is then exposed to acandidate compound that may function as a therapeutic agent for aTWEAK-related condition. Fibrotic, cardiac, kidney, liver, lung, skin,skeletal muscle, lipid, gastrointestinal tract, pancreas, reproductiveorgans, neural, cartilage, bone or connective tissue from the testanimal is then compared to the same tissue from a reference animal thatexpresses the exogenous DNA but has not been exposed to the compound;and it is determined whether the compound has affected any TWEAK-relatedcondition of the fibrotic, cardiac, kidney, liver, lung, skin, skeletalmuscle, lipid, gastrointestinal tract, pancreas, reproductive organs,neural, cartilage, bone or connective tissues.

In order to identify TWEAK agonist compounds that act as therapeuticagents for the treatment of TWEAK-related conditions according to thepresent invention, a test animal that either does or does not express anexogenous DNA encoding a TWEAK polypeptide, or a fragment, analog,mutein, or mimetic thereof may be exposed to a candidate compound thatmay function as a therapeutic agent for a TWEAK-related condition.Fibrotic, cardiac, kidney, liver, or lung tissue from the test animal isthen compared to the same tissue from a reference animal that has notbeen exposed to the compound; and it is determined whether the compoundhas induced any biological change in said tissues as described hereindue to TWEAK signaling in vivo.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the disclosure herein, including the appended claims.

The invention claimed is:
 1. A method for treating pulmonary, liver, orcardiac fibrosis in a human subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of aTWEAK antagonist, wherein the TWEAK antagonist is an antibody, or anantigen-binding fragment thereof, that binds human TWEAK.
 2. The methodof claim 1, wherein the fibrosis is liver fibrosis.
 3. The method ofclaim 1, wherein the fibrosis is cardiac fibrosis.
 4. The method ofclaim 1, wherein the fibrosis is pulmonary fibrosis.
 5. The method ofclaim 2, wherein the antibody or antigen-binding fragment thereof ishuman or humanized.
 6. The method of claim 2, wherein the antibody orantigen-binding fragment thereof is monoclonal or chimeric.
 7. Themethod of claim 2, wherein a full length antibody is administered. 8.The method of claim 2, wherein an antigen-binding fragment of theantibody is administered.
 9. The method of claim 2, wherein the TWEAKantagonist is administered to the subject via a route selected from thegroup consisting of: injection, transmucosal, oral, inhalation, ocular,rectal, stent implantation, topical, parenteral, long actingimplantation, sustained release, and aural routes.
 10. The method ofclaim 2, wherein the TWEAK antagonist is in a delivery formulationselected from the group consisting of: tablets, pills, liposomes,granules, spheres, dragees, capsules, liquids, gels, syrups, slurries,suspensions, stent coatings, and sustained-release formulations.
 11. Themethod of claim 3, wherein the antibody or antigen-binding fragmentthereof is human or humanized.
 12. The method of claim 3, wherein theantibody or antigen-binding fragment thereof is monoclonal or chimeric.13. The method of claim 3, wherein a full length antibody isadministered.
 14. The method of claim 3, wherein an antigen-bindingfragment of the antibody is administered.
 15. The method of claim 3,wherein the TWEAK antagonist is administered to the subject via a routeselected from the group consisting of: injection, transmucosal, oral,inhalation, ocular, rectal, stent implantation, topical, parenteral,long acting implantation, sustained release, and aural routes.
 16. Themethod of claim 3, wherein the TWEAK antagonist is in a deliveryformulation selected from the group consisting of: tablets, pills,liposomes, granules, spheres, dragees, capsules, liquids, gels, syrups,slurries, suspensions, stent coatings, and sustained-releaseformulations.
 17. The method of claim 4, wherein the antibody orantigen-binding fragment thereof is human or humanized.
 18. The methodof claim 4, wherein the antibody or antigen-binding fragment thereof ismonoclonal or chimeric.
 19. The method of claim 4, wherein a full lengthantibody is administered.
 20. The method of claim 4, wherein anantigen-binding fragment of the antibody is administered.
 21. The methodof claim 4, wherein the TWEAK antagonist is administered to the subjectvia a route selected from the group consisting of: injection,transmucosal, oral, inhalation, ocular, rectal, stent implantation,topical, parenteral, long acting implantation, sustained release, andaural routes.
 22. The method of claim 4, wherein the TWEAK antagonist isin a delivery formulation selected from the group consisting of:tablets, pills, liposomes, granules, spheres, dragees, capsules,liquids, gels, syrups, slurries, suspensions, stent coatings, andsustained-release formulations.